Random-primed reverse transcriptase-in vitro transcription method for rna amplification

ABSTRACT

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

1. TECHNICAL FIELD

[0001] The present invention relates to enzymatic amplification ofnucleic acids using two random primers, one of which contains a RNApolymerase promoter sequence, to generate a double stranded DNAtemplate, and in vitro transcription.

2. BACKGROUND OF THE INVENTION

[0002] The characterization of cellular gene expression findsapplication in a variety of disciplines, such as in the analysis ofdifferential expression between different tissue types, different stagesof cellular growth or between normal and diseased states. Recently,changes in gene expression have also been used to assess the activity ofnew drug candidates- and to identify new targets for drug development.The latter objective is accomplished by correlating the expression of agene or genes known to be affected by a particular drug with theexpression profile of other genes of unknown function when exposed tothat same drug; genes of unknown function that exhibit the same patternof regulation, or signature, in response to the drug are likely torepresent novel targets for pharmaceutical development. One particularlyuseful method of assaying gene expression at the level of transcriptionemploys DNA microarrays (Ramsay, Nature Biotechnol. 16: 40-44, 1998;Marshall and Hodgson, Nature Biotechnol. 16: 27-31, 1998; Lashkari etal., Proc. Natl. Acad. Sci. (USA) 94: 130-157, 1997; DeRisi et al.,Science 278: 680-6, 1997).

[0003] A number of methods for the amplification of nucleic acids havebeen described. Such methods include the “polymerase chain reaction”(PCR) (Mullis et al., U.S. Pat. No. 4,683,195), and a number oftranscription-based amplification methods (Malek et al., U.S. Pat. No.5,130,238; Kacian and Fultz, U.S. Pat. No. 5,399,491; Burg et al., U.S.Pat. No. 5,437,990). Each of these methods uses primer-dependent nucleicacid synthesis to generate a DNA or RNA product, which serves as atemplate for subsequent rounds of primer-dependent nucleic acidsynthesis. Each process uses (at least) two primer sequencescomplementary to different strands of a desired nucleic acid sequenceand results in an exponential increase in the number of copies of thetarget sequence. These amplification methods can provide enormousamplification (up to billion-fold). However, these methods havelimitations that make them not amenable for gene expression monitoringapplications. First, each process results in the specific amplificationof only the sequences that are bounded by the primer binding sites.Second, exponential amplification can introduce significant changes inthe relative amounts of specific target species—small differences in theyields of specific products (for example, due to differences in primerbinding efficiencies or enzyme processivity) become amplified with everysubsequent round of synthesis.

[0004] Amplification methods that utilize a primer containing a RNApolymerase promoter sequence (“promoter-primer”) are amenable to theamplification of heterogeneous mRNA populations. The vast majority ofmRNAs carry a homopolymer of 20-250 adenosine residues on their 3′ ends(the poly-A tail), and the use of poly-dT primers for cDNA synthesis isa fundamental tool of molecular biology. “Single-primer amplification”protocols have been reported (see e.g., Kacian et al., U.S. Pat. No.5,554,516; Van Gelder et al., U.S. Pat. No. 5,716,785). The methodsreported in these patents utilize a single promoter-primer containing aRNA polymerase promoter sequence and a sequence complementary to the3′-end of the desired nucleic acid target sequence(s). In both methods,the promoter-primer is added under conditions in which it hybridizes tothe target sequence(s) and is converted to a substrate for RNApolymerase. In both methods, the substrate intermediate is recognized byRNA polymerase, which produces multiple copies of RNA complementary tothe target sequence(s) (“antisense RNA”). Each method uses, or could beadapted to use, a primer containing poly-dT for amplification ofheterogeneous mRNA populations.

[0005] Amplification methods that proceed linearly during the course ofthe amplification reaction are less likely to introduce bias in therelative levels of different mRNAs than those that proceedexponentially. In the method described in Kacian et al., U.S. Pat. No.5,554,516, the amplification reaction contains a nucleic acid targetsequence, a promoter-primer, a RNA polymerase, a reverse transcriptase,and reagent and buffer conditions sufficient to allow amplification. Theamplification proceeds in a single tube under conditions of constanttemperature and ionic strength. Under these conditions, the antisenseRNA products of the reaction can serve as substrates for furtheramplification by non-specific priming and extension by the RNA-dependentDNA polymerase activity of reverse transcriptase. As such, theamplification described in U.S. Pat. No. 5,554,516 proceedsexponentially. In contrast, in specific examples described in Van Gelderet al., U.S. Pat. No. 5,716,785, cDNA synthesis and transcription occurin separation reactions separated by phenol/chloroform extraction andethanol precipitation (or dialysis), which may incidentally allow forthe amplification to proceed linearly since the RNA products cannotserve as substrates for further amplification.

[0006] The method described in U.S. Pat. No. 5,716,785 has been used toamplify cellular mRNA for gene expression monitoring (for example, R. N.Van Gelder et al. (1990), Proc. Natl. Acad. Sci. USA 87, 1663; D. J.Lockhart et al. (1996), Nature Biotechnol. 14, 1675). However, thisprocedure is not readily amenable to high throughput processing. Inpreferred embodiments of the method described in U.S. Pat. No.5,716,785, poly-A mRNA is primed with a promoter-primer containingpoly-dT and converted into double-stranded cDNA using a method describedby Gubler and Hoffman (U. Gubler and B. J. Hoffman (1983), Gene 25,263-269) and popularized by commercially available kits for cDNAsynthesis. Using this method for cDNA synthesis, first strand synthesisis performed using reverse transcriptase and second strand cDNA issynthesized using RNaseH and DNA polymerase I. After phenol/chloroformextraction and dialysis, double-stranded cDNA is transcribed by RNApolymerase to yield antisense RNA product. The phenol/chloroformextractions and buffer exchanges required in this procedure are laborintensive, and are not readily amenable to robotic handling.

[0007] A method of linear amplification of mRNA into antisense RNA hasbeen recently developed, U.S. Pat. No. 6,132,997 issued to Shannon(“Shannon”), which is incorporated by reference in its entirety for allpurposes. Shannon does not require a reverse transcriptase separationstep and is therefore readily amenable to high throughput processing.Shannon discloses a method in which mRNA is converted to cDNA(particularly double-stranded cDNA) using a promoter-primer having apoly-dT primer site linked to a promoter sequence so that the resultingcDNA is recognized by a RNA polymerase. The resultant cDNA is thentranscribed into RNA (particularly antisense RNA) in the presence of areverse transcriptase that is rendered incapable of RNA-dependent DNApolymerase activity during the transcription step.

[0008] A significant drawback of the Shannon method, however, is that itproduces a 3′ bias in the amplification of mRNA. Sequences that are morethan 1000 bp from the 3′ end to which the primer has hybridized areunderamplified with respect to sequences that are less than 1000 bp fromthe 3′ end, i.e., the sequences that are more than 1000 bp from the 3′end are amplified in less than linear amounts.

[0009] Thus there exists a need in the art for an improved method oflinear amplification of mRNA that is amenable to high throughputprocessing, that produces little or no 3′ bias, that improves theability to detect the 5′ ends of mRNA, and therefore achieves goodrepresentation of both the 3′ and 5′ regions of an original mRNA in theamplified complementary RNA (cRNA).

3. SUMMARY OF THE INVENTION

[0010] A random-primed reverse transcriptase-in vitro transcriptionmethod of linearly amplifying RNA is provided. According to the methodsof the invention, source RNA (or other single-stranded nucleic acid),preferably, mRNA, is converted to double-stranded cDNA using two randomprimers, one of which comprises a RNA polymerase promoter sequence(“promoter-primer”), to yield a double-stranded cDNA that comprises aRNA polymerase promoter that is recognized by a RNA polymerase.Preferably, the primer for first-strand cDNA synthesis is apromoter-primer and the primer for second-strand cDNA synthesis is not apromoter-primer. The double-stranded cDNA is then transcribed into RNAby the RNA polymerase, optimally in the presence of a reversetranscriptase that is rendered incapable of RNA-dependent DNA polymeraseactivity during this transcription step. The subject methods ofproducing linearly amplified RNA provide an improvement over priormethods in that little or no 3′ bias in the sequences of the nucleicacid population amplified is produced, and the ability to detect the 5′end sequences of the nucleic acids is improved. The methods of theinvention therefore achieve good representation of both the 3′ and 5′regions of the source nucleic acid in the amplified complementary RNA(cRNA). Linear amplification extents of at least 100-fold can beachieved using the subject methods. All of the benefits of linearamplification are achieved with the subject methods, such as theproduction of unbiased antisense RNA libraries from heterogeneous mRNAmixtures.

[0011] In particular, the invention provides a method for linearlyamplifying one or more single stranded nucleic acids, said methodcomprising (a) contacting said one or more single stranded nucleic acidswith a first set of oligonucleotides, each of which comprises a promotersequence and a sequence from a set of random sequences of at least 4nucleotides (but preferably 6 to 9 nucleotides, more preferably 9nucleotides), a second set of oligonucleotides, each of which comprises(preferably, consists of) of one or a set of random sequences of atleast 4 nucleotides (but preferably 6 to 9 nucleotides, more preferably6 nucleotides) and one or more enzymes that alone or in combinationcatalyze the synthesis of double-stranded cDNA, under conditionssuitable for the production of double-stranded cDNA; and (b) contactingthe double-stranded cDNA produced in step (a) with a RNA polymerase thatrecognizes said promoter sequence and ribonucleotides under conditionssuitable to effect transcription, thereby producing sense or antisenseRNA copies corresponding to said one or more single stranded nucleicacids. In a preferred embodiment, the second set of oligonucleotidesdoes not contain a promoter sequence. Alternatively, the cDNA may begenerated in two steps where the first step is the synthesis of firststrand cDNA using the first set of oligonucleotides and one or moreenzymes that catalyze first strand cDNA synthesis and the second step isthe synthesis of double-stranded cDNA by contacting the first strandcDNA made in the first step with the second set of oligonucleotides andone or more enzymes that alone or in combination catalzye second strandcDNA synthesis. In preferred embodiments, the enzyme used in step (a) isa reverse transcriptase. In an alternative embodiment, thesingle-stranded nucleic acid is also contacted in step (a) with apromoter-primer containing the same promoter sequence used in the set ofrandom primer-promoter primers used in step (a) and a polydT sequence ofat least 4 nucleotides (preferably at least 5 nucleotides, morepreferably 15 to 25 nucleotides, and most preferably 18 nucleotides).

[0012] The invention further provides kits for carrying out the linearamplification methods of the invention containing one or more componentsused in the methods of the inventions and instructions for use. In aparticular embodiment, the invention provides a kit for use in linearlyamplifying single stranded nucleic acids into sense or antisense RNA,said kit comprising a first set of oligonucleotides each comprising apromoter sequence and one of a set of random sequences of at least 4nucleotides; and a second set of oligonucleotides each of whichcomprises (preferably, consists of) of one of a set of random sequencesof at least four nucleotides. In a preferred embodiment the second setof oligonucleotides does not contain a promoter sequence In anotherembodiment, the kit also contains a reverse transcriptase and a RNApolymerase. In yet another embodiment, the kit further contains, inaddition to the two sets of random primers, oligonucleotides containingthe same promoter sequence as the random primer-promoter primeroligonucleotide and a polydT sequence of at least 5 nucleotides(preferably 18 nucleotides).

4. DESCRIPTION OF THE FIGURES.

[0013] FIGS. 1(A-B). Comparison of profiles obtained from single-geneanalysis using (A) the mRNA amplification method described in U.S. Pat.No. 6,132,997 (Shannon, issued Oct. 17, 2000) (“Shannon”) and (B) therandom-primed reverse transcriptase-in vitro transcription (RT-IVT)method of the invention. The graphs plot signal intensity (mlavg) ofoligonucleotides in a single gene (X-axis) as a function of the numberof base pairs from the 5′ end (Y-axis). The 3′ bias of signal intensityseen when the Shannon method is used cannot be seen when therandom-primed RT-IVT method is used, indicating that the random-primedRT-IVT method overcomes the 3′ bias of the Shannon method.

[0014]FIG. 2. Intensity difference as a function of distance from the 3′end. The graph shows the intensity of all oligonucleotides as a functionof distance from the 3′ end. The graph plots mlavg (Shannonmethod)−mlavg (random-primed RT-IVT method) (X-axis) versus log₁₀ of thenumber of bp from the 3′ end (Y-axis). The intensity obtained with theShannon method is greater than the intensity obtained with therandom-primed RT-IVT method for probes less than 1000 bp from the 3′ endof the message. The intensity obtained with the Shannon method is lessthan the intensity obtained with the random-primed RT-IVT method forprobes greater than 1000 bp from the 3′ end of the message.

[0015] FIGS. 3(A-C). Signature differences in the numbers andpercentages of significant data points. The top graph (A) plots thenumber of probes (X-axis) versus the log₁₀ (bp) (Y-axis). The middlegraph (B) plots the number of signatures (X-axis) versus the log₁₀ (bp)(Y-axis). The bottom graph (C) plots the fraction of signatures versusthe log₁₀ (bp) (Y-axis). As can be seen in the bottom graph, therandom-primed RT-IVT method outcompetes the Shannon method for probesgreater than 1000 bp from the 3′ end. Note the black arrow atapproximately 700 bp where random-primed RT-IVT method is morerepresentative than the Shannon method. Stars: Shannon method. Circles:random-primed RT-IVT method.

[0016] FIGS. 4(A-C). Shows the results obtained when the amplificationmethods of the invention were run using a primer comprising a T7 RNApolymerase promoter site and an poly-dT₁₈ sequence (“T7-dT₁₈”), inaddition to using random T7-dN₉ and dN₆ primers. The top graph (A) plotsthe number of probes (X-axis) versus the log₁₀ (bp) (Y-axis). The middlegraph (B) plots the number of signatures (X-axis) versus the log₁₀ (bp)(Y-axis). The bottom graph (C) plots the fraction (“frac”) or percentageof signatures versus the log₁₀ (bp) (Y-axis). As can be seen in thebottom graph, the number of probes at greater than 1000 base pairs isgreater with the random-primed RT-IVT method. Using both the T7-dT₁₈ andrandom T7-dN₉ primers for first strand cDNA synthesis improves thefraction of statistically significant probes more efficiently thaneither the Shannon method or the method of the invention in which justthe random T7-dN₉ primer is used. Stars: Shannon method. Circles:random-primed RT-IVT method.

5. DETAILED DESCRIPTION OF THE INVENTION

[0017] A random-primed reverse transcriptase-in vitro transcription(RT-IVT) method of linearly amplifying RNA is provided. According to themethods of the invention, source RNA (or other single-stranded nucleicacid), preferably, mRNA, is converted to double-stranded cDNA using tworandom primers, one of which comprises a RNA polymerase promotersequence (“promoter-primer”), to yield a double-stranded cDNA thatcomprises a RNA polymerase promoter that is recognized by a RNApolymerase. Thus, “promoter sequence” refers to a single-strandednucleotide sequence that when double-stranded (i.e., paired with itsreverse-complement) forms a RNA polymerase promoter that is recognizedby a RNA polymerase. Preferably, the primer for first-strand cDNAsynthesis is a promoter-primer and the primer for second-strand cDNAsynthesis is not a promoter-primer. Optionally, the cDNA synthesisreaction contains a mixture of the random-sequence-promoter primer andan oligonucleotide containing the promoter sequence and an oligodTsequence. The double-stranded cDNA is then transcribed into RNA by theRNA polymerase, optimally in the presence of a reverse transcriptasethat is rendered incapable of RNA-dependent DNA polymerase activityduring this transcription step.

[0018] The subject methods of producing linearly amplified RNA providean improvement over prior methods in that little or no 3′ bias in thesequences of the nucleic acid population amplified is produced, and theability to detect the 5′ end sequences of the nucleic acids is improved.The methods of the invention therefore achieve good representation ofboth the 3′ and 5′ regions of the source nucleic acid in the amplifiedcomplementary RNA (cRNA). Linear amplification extents of at least100-fold can be achieved using the subject methods. All of the benefitsof linear amplification are achieved with the subject methods, such asthe production of unbiased antisense RNA libraries from heterogeneousmRNA mixtures.

[0019] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the subsectionsset forth below.

[0020] 5.1. Methods of Nucleic Acid Amplification

[0021] The invention provides methods for producing amplified amounts ofeither sense or antisense RNA from an initial amount of sourcesingle-stranded nucleic acid, preferably poly-A⁺ RNA or mRNA. Byamplified amounts is meant that for each initial source of nucleic acid,multiple corresponding sense or antisense RNAs are produced. The termantisense RNA is defined here as RNA complementary to the sourcesingle-stranded nucleic acid. By corresponding is meant that the senseor antisense RNA shares a substantial sequence identity with thesequence of, or the sequence complementary to (i.e., the complement ofthe initial source nucleic acid), the source nucleic acid. Substantialsequence identity means at least 95%, usually at least 98%, and moreusually at least 99%, and, in certain embodiments, 100% sequenceidentity, where sequence identity is determined using the BLASTalgorithm, as described in Altschul et al. (1990), J. Mol. Biol.215:403-410 (using the published default setting, i.e., parameters w=4,t=17). Generally, the number of corresponding antisense RNA moleculesproduced for each initial nucleic acid during the subject linearamplification methods will be at least about 10, usually at least about50, more usually at least about 100, and may be as great as 600 orgreater, but often does not exceed about 1000.

[0022] The subject methods can be used to produce amplified amounts ofRNA corresponding to substantially all of the nucleic acid present inthe initial sample, or to a proportion or fraction of the total numberof distinct nucleic acids present in the initial sample. Bysubstantially all of the nucleic acid present in the sample is meantmore than 90%, usually more than 95%, where that portion not amplifiedis solely the result of inefficiencies of the reaction and notintentionally excluded from amplification.

[0023] In a specific embodiment, only a single cycle of reversetranscription is carried out. In alternative embodiments, more than onecycle of reverse transcription is performed (with transcription anddenaturation between cycles). For example, in a specific embodiment, afirst cycle of reverse transcription is carried out wherein one or moresingle stranded nucleic acids are (a) contacted with a first set ofoligonucleotides, each of which comprises a promoter sequence and asequence from a set of random sequences of at least 4 nucleotides (butpreferably 6 to 9 nucleotides, more preferably 9 nucleotides), a secondset of oligonucleotides, each of which comprises (preferably, consistsof) of one or a set of random sequences of at least 4 nucleotides (butpreferably 6 to 9 nucleotides, more preferably 6 nucleotides) and one ormore enzymes that alone or in combination catalyze the synthesis ofdouble-stranded cDNA, under conditions suitable for the production ofdouble-stranded cDNA. The resultant double-stranded cDNA is then (b)contacted with a RNA polymerase that recognizes said promoter sequenceand ribonucleotides under conditions suitable to effect transcription(i.e., in vitro transcription or “IVT”), thereby producing sense orantisense RNA copies corresponding to said one or more single strandednucleic acids. The resultant sense or antisense RNA copies are thenreverse transcribed in a second cycle of reverse transcription, asdescribed in step (a) above, and the resultant double-stranded cDNA isthen transcribed via IVT into sense or antisense RNA copies as describedin step (b) above. Additional cycles of RT-IVT may be performed toobtain the desired quantity of sense or antisense RNA copies.

[0024] According to the methods of the invention, additional linearamplification is afforded by a subsequent in vitro transcription (IVT)step as described below in Section 5.2. During IVT, the double-strandedcDNA produced in the first step is transcribed by RNA polymerase toyield RNA that is complementary to the initial RNA target from which itis amplified. This combination of cDNA synthesis and IVT enables thegeneration of a relatively large amount of cRNA from a very smallstarting amount of nucleic acid without loss of fidelity, andparticularly, without 3′ amplification bias.

[0025] In one embodiment of the invention (see Example 1, Section 6),0.2 μg (200 ng) of source mRNA is used.

[0026] In another embodiment of the invention, nucleic acidamplification is performed in situ, on samples of preserved or freshcells or tissues (see, e.g., Nuovo, 1997, PCR In Situ Hybridization:Protocols and Applications, Third Edition, Lippincott-Raven Press, NewYork).

[0027] The subject methods may be applied to other amplification systemsin which an oligonucleotide is incorporated into an amplificationproduct such as polymerase chain reaction (PCR) systems (U.S. Pat. No.4,683,195, Mullis et al., entitled “Process for amplifying, detecting,and/or-cloning nucleic acid sequences,” issued Jul. 28, 1987; U.S. Pat.No. 4,683,202, Mullis, entitled “Process for amplifying nucleic acidsequences,” issued Jul. 28, 1987)

[0028] 5.1.1. cDNA Synthesis

[0029] Double-stranded cDNA molecules can be synthesized from acollection of RNAs (or other single-stranded nucleic acids), e.g., mRNAspresent in a population of cells, by methods well-known in the art. Inorder for the cDNAs produced in this step to be useful in the methods ofthe invention, it is necessary to incorporate a RNA polymerase promoterinto the cDNA molecules during synthesis. This enables the cDNAmolecules to serve as templates for RNA transcription. This isaccomplished by choosing one or more primers for the cDNA synthesisreaction that comprise a single-stranded, synthetic oligonucleotidecontaining a RNA polymerase promoter sequence in sense orientation. This“promoter-primer” may be used to prime either first strand and/or secondstrand cDNA synthesis. In preferred embodiments, the “promoter-primer”primes first strand cDNA synthesis and the promoter is in theappropriate orientation to promote synthesis of antisense RNA.

[0030] Typically, only one RNA polymerase promoter sequence-containingprimer is used during cDNA synthesis. Preferably, the promoter-primer isused to prime first strand cDNA synthesis. Following reversetranscription, the resultant RNA polymerase promoter-containingdouble-stranded cDNA is transcribed into RNA using a RNA polymerasecapable of binding to the RNA polymerase promoter introduced during cDNAsynthesis (see below Section 5.2).

[0031] In a preferred embodiment, the primer for first strand cDNAsynthesis is a mixture of random primers linked to a promoter sequencethat prime synthesis in a direction toward the 5′ end of the nucleicacids (e.g., mRNAs) in the sample, and the primer for second strand cDNAsynthesis is a mixture of random primers that prime synthesis ofdouble-stranded cDNA from substantially all the first strand cDNAs thusproduced.

[0032] Preferably, the first-strand primer is a random promoter-primer,wherein the random (poly-dN) sequence is operably linked to a RNApolymerase promoter sequence. In one aspect, the first-strand primer isa mixture of primers, each primer comprising a RNA polymerase promotersequence and a 3′ end or 3′ distal sequence of 6-9 nucleotides,preferably 9 nucleotides. The mixture of primers comprises randomprimers, i.e., primers having an A, a G, a C, or a T residue present ineach position of the 3′ end sequence or 3′ distal sequence (i.e., thenon-promoter sequence). In particular, the random primer for primingfirst strand cDNA synthesis is a random promoter-primer that includes:(a) a poly-dN region for hybridization to the mRNA; and (b) a RNApolymerase promoter region 5′ of the poly-dN region that is in anorientation capable of directing transcription of antisense RNA when itprimes first strand cDNA synthesis. The poly-dN region is sufficientlylong to provide for efficient hybridization to the mRNA, where theregion typically ranges in length from 4-50 nucleotides in length,preferably 6-25 nucleotides in length, more preferably from 6-12, andmost preferably, 9 nucleotides in length, i.e., a random 9-mer. Inspecific embodiments, the poly-dN region is 4, 5, 6, 7, 8, 9, 10, 11, or12 nucleotides in length.

[0033] In a preferred embodiment, the random promoter-primer used toprime first strand cDNA synthesis is a random 9-mer operably linked to aT7 RNA polymerase promoter sequence (T7-dN₉: (5′) AAT TAA TAC GAC TCACTA TAG GGA GAT NNN NNN NNN (3′) (N=A, T, C or G) (SEQ ID NO.: 1)).

[0034] In another embodiment, the random promoter-primers used to primefirst strand cDNA synthesis are a complete set of all (or almost all)combinations of random 9-mers, i.e., a total of 4⁹ 9-mers, linked to aT7 RNA polymerase promoter sequence.

[0035] In another embodiment, a poly-dT primer comprising a RNApolymerase promoter sequence and a random dN primer comprising a RNApolymerase promoter sequence are used together to prime first strandcDNA synthesis. Preferably, the poly-dT-promoter primer and the randomprimer-promoter primer contain the same promoter sequence. In particularembodiments the poly-dT sequence is at least 5 thymidilate residues,preferably 15 to 25 residues and, preferably 18 residues. In a preferredembodiment, a T7-dT₁₈ primer and a T7-dN₉ primer are used to prime firststrand cDNA synthesis.

[0036] A number of RNA polymerase promoters may be used for the promoterregion of the promoter-primer. Suitable promoter regions will be capableof initiating transcription from an operably linked DNA sequence in thepresence of ribonucleotides and a RNA polymerase under suitableconditions. The term “operably linked” refers to a functional linkage,i.e., the promoter will be linked in an orientation to permittranscription of sense or antisense RNA. Preferably the linkage iscovalent, most preferably by a nucleotide bond. Most preferably, thepromoter is linked in an orientation to permit transcription ofantisense RNA when the promoter is incorporated into the first strand ofcDNA synthesis. A linker oligonucleotide between the promoter and theDNA may be present, and if, present, will typically comprise betweenabout 5 and 20 bases, but may be smaller or larger as desired. Thepromoter region is of sufficient length to promote transcription, andwill usually comprise between about 15 and 250 nucleotides, preferablybetween about 17 and 60 nucleotides, from a naturally occurring RNApolymerase promoter or a consensus promoter region, as described inAlberts et al. (1989) in Molecular Biology of the Cell, 2d Ed. (GarlandPublishing, Inc.), or any other variant that promotes transcription. Ina specific embodiment, the promoter region is 36 nucleotides. Preferredpromoter regions include the bacteriophage SP6 and T3 promoters and,most preferably, T7 promoters.

[0037] The random promoter-primer and/or the random primer mayadditionally contain a restriction site, in the middle or at the 5′distal end of the primer, but preferably not immediately at the 5′terminus. The restriction site may be used for cloning in to a vector.Restriction enzymes and the sites they recognize can be found, forexample, in Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual(2nd Ed.), Vol. 1, Chapter 5, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

[0038] The primers of the invention may be prepared using any suitablemethod known in the art, e.g., as described in Section 5.3 infra.

[0039] Preferably both first- and second-strand cDNA synthesis isproduced by reverse transcription, wherein DNA is made from RNA usingthe enzyme reverse transcriptase. Reverse transcriptase is found in allretroviruses and is commonly obtained from avian myeloblastoma virus orMoloney murine leukemia virus; enzyme from these sources is commerciallyavailable from Life Technologies (Gaithersburg, Md.) and BoehringerMannheim (Indianapolis, Ind.).

[0040] The catalytic activities required to convert thepromoter-primer-mRNA hybrid to double-stranded cDNA are a RNA-dependentDNA polymerase activity, a RNaseH activity, and a DNA-dependent DNApolymerase activity. Most reverse transcriptases, including thosederived from Moloney murine leukemia virus (MMLV-RT), avianmyeloblastosis virus (AMV-RT), bovine leukemia virus (BLV-RT), Roussarcoma virus (RSV) and human immunodeficiency virus (HIV-RT) catalyzeeach of these activities. These reverse transcriptases are sufficient toconvert a primer-mRNA hybrid to double-stranded DNA in the presence ofadditional reagents that include, but are not limited to: dNTPs;monovalent and divalent cations, e.g., KCl, MgCl₂; sulfhydryl reagents,e.g., dithiothreitol; and buffering agents, e.g., Tris-Cl.Alternatively, a variety of proteins that catalyze one or two of theseactivities can be added to the cDNA synthesis reaction. For example,MMLV reverse transcriptase lacking RNaseH activity (described in U.S.Pat. No. 5,405,776) catalyzes RNA-dependent DNA polymerase activity andDNA-dependent DNA polymerase activity. These proteins may be addedtogether during a single reaction step, or added sequentially during twoor more substeps. Preferably, MMLV is used for both first- andsecond-strand cDNA synthesis. As described above, preferably the reversetranscriptase is inactivated prior to or inhibited during thetranscription step of the method.

[0041] In general, it is preferable for the RNA-containing sample tocontain purified poly-A⁺ RNA (mRNA). In one embodiment, a randompromoter-primer is hybridized with an initial mRNA (poly-A⁺ RNA) sample.The promoter-primer is contacted with the mRNA under conditions thatallow the poly-dN site to hybridize to the mRNA. The randompromoter-primer-mRNA hybrid is then converted to a double-stranded cDNAproduct that is recognized by a RNA polymerase.

[0042] In a preferred embodiment, first-strand cDNA synthesis is allowedto proceed at a lower temperature (for example, 25° C.) for a certainperiod of time (e.g., 10 min) prior to increasing the temperature (e.g.,to 40° C.) for the remainder of the reverse transcription reaction,which improves annealing of the first primer (e.g., the promoter-primer)to its target nucleic acid sequence.

[0043] In the subject methods, conversion of the primer-mRNA hybrid todouble-stranded cDNA proceeds by priming second strand cDNA synthesiswith a random primer in the presence of a DNA-dependent DNA polymeraseactivity.

[0044] In another embodiment, the primer for second strand cDNAsynthesis is a mixture of primers consisting of a poly-dN sequence thatis sufficiently long to provide for efficient hybridization to the mRNA.The sequence typically ranges in length from 4-50, preferably 6-25, morepreferably 6-12 or 6-9 and most preferably 6 degenerate bases, i.e., arandom hexamer (dN₆), wherein the degenerate bases may be A, T, G, or C.(In theory, the primer should hybridize on average 4⁶ or 4096 base pairsfrom the 3′ priming site of the first-strand cDNA.) In specificembodiments, the poly-dN sequence is 4, 5, 6, 7, 8, 9, 10, 11, or 12nucleotides in length.

[0045] In a specific embodiment, the random primers used to prime secondstrand cDNA synthesis will be a complete set of all combinations ofrandom hexamers, i.e., a total of 4⁶ or 4096 hexamers.

[0046] Additional proteins that may enhance the yield of double-strandedDNA products may also be added to the cDNA synthesis reaction. Theseproteins include a variety of DNA polymerases (such as those derivedfrom E. coli, thermophilic bacteria, archaebacteria, phage, yeasts,Neurosporas, Drosophilas, primates and rodents), and DNA ligases (suchas those derived from phage or cellular sources, including T4 DNA ligaseand E. coli DNA ligase).

[0047] The second strand cDNA synthesis results in the creation of adouble-stranded promoter region. The second strand cDNA includes notonly a sequence of nucleotide residues that comprise a DNA copy of themRNA template, but also additional sequences at its 3′ end that arecomplementary to the promoter-primer used to prime first strand cDNAsynthesis. The double-stranded promoter region serves as a recognitionsite and transcription initiation site for RNA polymerase, which usesthe second strand cDNA as a template for multiple rounds of RNAsynthesis during the next stage of the subject methods (see Section 5.2,“Transcription of cDNA,” below).

[0048] Depending on the particular protocol, the same or different DNApolymerases may be employed during the cDNA synthesis step. In apreferred embodiment, a single reverse transcriptase, most preferablyMMLV-RT, is used as a source of all the requisite activities necessaryto convert the primer-mRNA hybrid to double-stranded cDNA. In anotherpreferred embodiment, the polymerase employed in first strand cDNAsynthesis is different from that which is employed in second strand cDNAsynthesis. Specifically, a reverse transcriptase lacking RNaseH activity(e.g., SUPERSCRIPT II™) is combined with the primer-mRNA hybrid during afirst substep for first strand synthesis. A source of RNaseH activity,such as E. coli RNaseH or MMLV-RT, but most preferably MMLV-RT, is addedduring a second substep to initiate second strand synthesis.

[0049] In yet other embodiments, the requisite-activities are providedby a plurality of distinct enzymes. The manner in which double-strandedcDNA is produced from the initial mRNA is not critical to certainembodiments of the invention. However, the preferred embodiments useMMLV-RT, or a combination of SUPERSCRIPT II™ and MMLV-RT, or acombination of SUPERSCRIPT II™ and E. coli RNaseH, for cDNA synthesis asthese embodiments yield certain desired results. Specifically, in thepreferred embodiments, reaction conditions were chosen so that enzymespresent during the cDNA synthesis do not adversely affect the subsequenttranscription reaction. Potential inhibitors include, but are notlimited to, RNase contaminants of certain enzyme preparations.

[0050] 5.2. Transcription of cDNA

[0051] The next step of the subject method is the preparation of RNAfrom the double-stranded cDNA prepared in the first step. During thisstep, the double-stranded cDNA produced in the first step is transcribedby RNA polymerase to yield RNA that, in certain embodiments, iscomplementary to the initial nucleic acid target from which it isamplified. This step is sometimes referred to as “in vitrotranscription” (IVT).

[0052] The promoter regions that find use in the methods of theinvention are regions where RNA polymerase binds tightly to the DNA andcontain the start site and signal for RNA synthesis to begin. A widevariety of promoters are known and many are very well characterized. Ingeneral, prokaryotic promoters are preferred over eukaryotic promoters,and phage or virus promoters most preferred. The RNA polymerase promotersequence is therefore preferably derived from a prokaryote such as E.coli or the bacteriophage T7, SP6, and T3, with the T7 RNA polymerasepromoter sequence particularly preferred. T7, T3 and SP6 promoterregions are described in Chamberlin and Ryan, The Enzymes (ed. P. Boyer,Academic Press, New York) (1982) pp 87-108, which excerpt is herebyincorporated by reference in its entirety.

[0053] The RNA polymerase used for transcription must be capable ofbinding to the particular RNA polymerase promoter sequence contained inthe primer; hence usually the RNA polymerase promoter sequence and thepolymerase will be homologous. For example, if the T7 RNA polymerasepromoter sequence is employed in the primer, it is preferred to use T7RNA polymerase to drive transcription. T7 polymerase is commerciallyavailable from several sources, including Promega Biotech (Madison,Wis.) and Epicenter Technologies (Madison, Wis.).

[0054] In a preferred embodiment, the random promoter-primer used toprime first strand cDNA synthesis comprises a T7 promoter sequence-dN₉,and the RNA polymerase employed is T7 RNA polymerase.

[0055] Preferably, the RNA polymerase promoter sequence is located at ornear the 5′ terminus of the primer, in an orientation permittingtranscription of the RNA population under study.

[0056] For this transcription step, the presence of the RNA polymerasepromoter region on the double-stranded cDNA is exploited for theproduction of sense and/or antisense RNA. To synthesize the RNA, thedouble-stranded DNA is contacted with the appropriate RNA polymerase inthe presence of the four ribonucleotides, under conditions sufficientfor RNA transcription to occur.

[0057] In one embodiment, the conditions for RNA transcription are thosedescribed in Section 6, Example 1. Briefly, the transcription mix andthe transcription reaction are as follows. 60 μl of Transcription Mixare aliquoted into each sample tube. The transcription reactions areincubated at 40° C. for 16 hrs. Transcription Mix Component Volume (μl)Nuclease-free water 22.8 5x Transcription Buffer 16 100 mM DTT 6.0 NTPs(25 mM A, G, C, 6.0 mM UTP) 8.0 aa UTP (allylamine-derivatized UTP) 2.0(75 mM) 200 mM MgCl₂ 3.3 RNAGuard ™, Pharmacia (36 U/μl) 0.5 InorganicPyrophosphatase (200 U/ml) 0.6 T7 RNA polymerase (2500 U/μl) 0.8 Volumeof Transcription Mix 60

[0058] Composition of Transcription Reaction Final concentrationComponent or amount Double-strand cDNA Approximately 400 ng Tris-HCl, pH7.5 52 mM MgCl₂ 15 mM KCl 19 mM NaCl 10 mM Spermidine 2 mM DTT 10 mMATP, GTP, CTP 2.5 mM each UTP 0.6 mM aa UTP 1.9 mM T7 RNA polymerase2000 U RNAGuard ™, Pharmacia 18 U Inorganic pyrophosphatase 0.12 U Totalreaction volume 80 μl

[0059] Other suitable conditions for RNA transcription using RNApolymerases are known in the art, see e.g., Milligan and Uhlenbeck(1989), Methods in Enzymol. 180, 51 (which is hereby incorporated byreference in its entirety).

[0060] In one aspect of the invention, the transcription step is carriedout in the presence of reverse transcriptase that is present in thereaction mixture from the double-stranded cDNA synthesis. Thus, thesubject methods do not involve a step in which the double-stranded cDNAis physically separated from the reverse transcriptase followingdouble-stranded cDNA preparation facilitating high throughputamplification and analysis. In this aspect of the invention, the reversetranscriptase that is present during the transcription step is renderedinactive. Thus, the transcription step is carried out in the presence ofa reverse transcriptase that is unable to catalyze RNA-dependent DNApolymerase activity, at least for the duration of the transcriptionstep. As a result, the RNA products of the transcription reaction cannotserve as substrates for additional rounds of cDNA synthesis, and theamplification process cannot proceed exponentially.

[0061] The reverse transcriptase present during the transcription stepmay be rendered inactive using any convenient protocol. Thetranscriptase may be irreversibly or reversibly rendered inactive. Wherethe transcriptase is reversibly rendered inactive, the transcriptase isphysically or chemically altered so as to no longer be able to catalyzeRNA-dependent DNA polymerase activity. The transcriptase may beirreversibly inactivated by any convenient means. Thus, the reversetranscriptase may be heat inactivated, in which the reaction mixture issubjected to heating to a temperature sufficient to inactivate thereverse transcriptase prior to commencement of the transcription step.In these embodiments, the temperature of the reaction mixture andtherefore the reverse transcriptase present therein is typically raisedto 55° C. to 70° C. for 5 to 60 minutes, usually to about 65° C. for 15to 20 minutes.

[0062] Alternatively, reverse transcriptase may be irreversiblyinactivated by introducing a reagent into the reaction mixture thatchemically alters the enzyme so that it no longer has RNA-dependent DNApolymerase activity. In yet other embodiments, the reverse transcriptaseis reversibly inactivated. In these embodiments, the transcription stepmay be carried out in the presence of an inhibitor of RNA-dependent DNApolymerase activity. Any convenient reverse transcriptase inhibitor maybe employed that is capable of inhibiting RNA-dependent DNA polymeraseactivity a sufficient amount to provide for linear amplification.However, these inhibitors should not adversely affect RNA polymeraseactivity. Reverse transcriptase inhibitors of interest include ddNTPs,such as ddATP, ddCTP, ddGTP or ddTTP, or a combination thereof, thetotal concentration of the inhibitor typically ranges from about 50 μMto 200 μM.

[0063] Because of the nature of the subject methods, all of thenecessary polymerization reactions, i.e., first strand cDNA synthesis,second strand cDNA synthesis and RNA transcription, may be carried outin the same reaction vessel at the same temperature, such thattemperature cycling is not required. As such, the subject methods areparticularly suited for automation, as the requisite reagents for eachof the above steps need merely be added to the reaction mixture in thereaction vessel, without any complicated separation steps beingperformed, such as phenol/chloroform extraction. A further feature ofthe subject invention is that, despite its simplicity, it yields highamplification extents, where the amplification extents (mass of RNAproduct/mass of nucleic acid target) typically are at least about50-fold, usually at least about 200-fold and may be as high as 600-foldor higher. Furthermore, such amplification extents are achieved with lowvariability, e.g., coefficients of variation about the meanamplification extents that do not exceed about 10%, and usually do notexceed about 5%.

[0064] The resultant cRNA (particularly antisense RNA) produced by thesubject methods finds use in a variety of applications. RNA amplified bythe methods of the invention may be labeled and employed to profile geneexpression in different populations of cells. In a preferred embodiment,the amplified RNA is used for quantitative comparisons of geneexpression between different populations of cells or between populationsof cells exposed to different stimuli. For example, the resultantantisense RNA can be used in expression profiling analysis on suchplatforms as DNA microarrays, for construction of “driver” forsubtractive hybridization assays, for cDNA library construction, and thelike. Especially facilitated by the subject methods are studies ofdifferential gene expression in mammalian cells or cell populations. Thecells may be from blood (e.g., white cells, such as T or B cells) orfrom tissue derived from solid organs, such as brain, spleen, bone,heart, vascular, lung, kidney, liver, pituitary, endocrine glands, lymphnode, dispersed primary cells, tumor cells, or the like.

[0065] The RNA amplification technology can also be applied to improvemethods of detecting and isolating nucleic acid sequences that vary inabundance among different populations using the technique known assubtractive hybridization. In such assays, two nucleic acid populations,one sense and the other antisense, are allowed to mix with one anotherwith one population being present in molar excess (“driver”). Underappropriate conditions, the sequences represented in both populationsform hybrids, whereas sequences present in only one population remainsingle-stranded. Thereafter, various well known techniques are used toseparate the unhybridized molecules representing differentiallyexpressed sequences. The amplification technology described herein maybe used to construct large amounts of antisense RNA for use as “driver”in such experiments.

[0066] 5.3. Oligonucleotides

[0067] A primer may be prepared by any suitable method, such asphosphotriester and phosphodiester methods of synthesis, or automatedembodiments thereof. It is also possible to use a primer that has beenisolated from a biological source, such as a restriction endonucleasedigest, although a synthetic primer is preferred.

[0068] An oligonucleotide primer can be DNA, RNA, chimeric mixtures orderivatives or modified versions thereof, so long as it is still capableof priming the desired reaction. The oligonucleotide primer can bemodified at the base moiety, sugar moiety, or phosphate backbone, andmay include other appending groups or labels, so long as it is stillcapable of priming the desired amplification reaction.

[0069] For example, an oligonucleotide primer may comprise at least onemodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

[0070] In another embodiment, the oligonucleotide primer comprises atleast one modified sugar moiety selected from the group including butnot limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0071] In yet another embodiment, the oligonucleotide primer comprisesat least one modified phosphate backbone selected from the groupconsisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0072] An oligonucleotide primer for use in the methods of the inventionmay be derived by cleavage of a larger nucleic acid fragment usingnon-specific nucleic acid cleaving chemicals or enzymes or site-specificrestriction endonucleases; or by synthesis by standard methods known inthe art, e.g., by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.) andstandard phosphoramidite chemistry. As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al. (1988,Nucl. Acids Res. 16:3209-3221), methylphosphonate oligonucleotides canbe prepared by use of controlled pore glass polymer supports (Sarin etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

[0073] Once the desired oligonucleotide is synthesized, it is cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present. Theoligonucleotide may then be purified by any method known in the art,including extraction and gel purification. The concentration and purityof the oligonucleotide may be determined by examining oligonucleotidethat has been separated on an acrylamide gel, or by measuring theoptical density at 260 nm in a spectrophotometer.

[0074] 5.4. Methods of Labeling of Nucleic Acid Amplification Products

[0075] Nucleic acid amplification products such as amplified RNA may belabeled with any art-known detectable marker, including radioactivelabels such as ³²P, ³⁵S, ³H, and the like; fluorophores;chemiluminescers; or enzymatic markers. In a preferred embodiment, thelabel is fluorescent. Exemplary suitable fluorophore moieties that canbe selected as labels are listed in Table 1. TABLE 1 Suitablefluorophore moieties that can be selected as labels4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS)-(4-anilino-1-naphthyl)maleimide anthranilamide BrilliantYellow coumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcoumarin (Coumarin 151) Cy3 Cy5cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylarnino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate Reactive Red 4 (Cibacron ® Brilliant Red3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX)6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 110 rhodamine 123 rhodamine Xisothiocyanate sulforhodamine B sulforhodamine 101 sulfonyl chloridederivative of sulforhodamine 101 (Texas Red)N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodaminetetramethyl rhodamine isothiocyanate (TRITC) riboflavin rosolic acidterbium chelate derivatives

[0076] 5.4.1. Labeling of RNA

[0077] Depending on the particular intended use of the RNA amplificationproducts, the RNA amplification products may be labeled. The RNA may belabeled with any art-known detectable marker, including but not limitedto radioactive labels such as ³²P, ³⁵S, ³H, and the like; fluorophores;chemiluminescers; or enzymatic markers (e.g., as listed in Table 1).

[0078] Labeling of RNA is preferably accomplished by including one ormore labeled NTPs in the in vitro transcription (IVT) reaction mixture.NTPs may be directly labeled with a radioisotope, such as ³²P, ³⁵S, ³H;radiolabeled NTPs are available from several sources, including NewEngland Nuclear (Boston, Mass.) and Amersham. NTPs may be directlylabeled with a fluorescent label such as Cy3 or Cy5. In one embodiment,biotinylated or allylamine-derivatized NTPs are incorporated during theIVT reaction and the resultant cRNAs are thereafter labeled, forexample, by the addition of fluorophore-conjugated avidin, in the caseof biotin, or the NHS ester of a fluorophore, in the case of allylamine.In another embodiment, fluorescently labeled NTPs may be incorporatedduring the IVT reaction, which fluorescently labels the resultant cRNAsdirectly.

[0079] RNA may be fluorescently labeled with fluorescently taggednucleotides (e.g., fluorescently labeled ATP, UTP, GTP or CTP) that areincorporated into the antisense RNA product during the transcriptionstep. Fluorescent moieties that may be used to tag nucleotides forproducing labeled antisense RNA include: fluorescein, the cyanine dyes,such as Cy3, Cy5, Alexa 542, Bodipy 630/650, and the like. Other labelsmay also be employed as are known in the art. Exemplary fluorophoremoieties that can be used as labels are listed in Table 1. The preferredlabel in the subject methods is a fluorophore, such as fluoresceinisothiocyanate, lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5particularly preferred.

[0080] Not only fluorophores, but also chemiluminescers and enzymes,among others, may be used as labels. In yet another embodiment, the RNAis labeled with an enzymatic marker that produces a detectable signalwhen a particular chemical reaction is conducted, such as alkalinephosphatase or horseradish peroxidase. Such enzymatic markers arepreferably heat stable, so as to survive the denaturing steps of theamplification process.

[0081] RNA may also be indirectly labeled by incorporating a nucleotidelinked covalently to a hapten or to a molecule such as biotin, to whicha labeled avidin molecule may be bound, or digoxygenin, to which alabeled anti-digoxygenin antibody may be bound. RNA may be labeled withlabeling moieties during chemical synthesis or the label may be attachedafter synthesis by methods known in the art.

[0082] Labeling of RNA is preferably accomplished by preparing cRNA thatis fluorescently labeled with NHS-esters. Most preferably, labeling ofRNA is accomplished in a two-step procedure in whichallylamine-derivatized UTP (aa UTP) is incorporated during IVT.Following the IVT reaction, unincorporated nucleotides are removed andthe allylamine-containing RNAs are conjugated to theN-hydroxysuccinimide (NHS) esters of Cy3 or Cy5.

[0083] In a preferred embodiment, 5-(3-Aminoallyl)uridine5′-triphosphate is incorporated into the RNA amplification productduring transcription and post-synthetically coupled to Cy-NHS, eitherCy3-NHS or Cy5-NHS.

[0084] In a specific embodiment, a two-step method of preparingfluorescent-labeled cRNA may be used in two color hybridizations to DNAmicroarrays. Such a two-step method is disclosed in U.S. Ser. No.09/411,074, filed Oct. 4, 1999, the disclosure of which is hereinincorporated by reference. In one embodiment, aminoallyl (aa)-labelednucleic acids are prepared by incorporation of aa-nucleotides. aa-UTP(Sigma A-5660) may be used for labeling cRNA. aa-cRNA is prepared usingthe Ambion MegaScript T7 RNA polymerase in vitro transcription kit, withaa-UTP substituted at 50-100% of the total UTP concentration. It isessential to remove all traces of amine-containing buffers such as Trisprior to derivatizing the aa-nucleic acids. aa-Nucleic acids prepared inenzymatic reactions are preferably cleaned up on appropriate QIAGENcolumns: RNeasy® Mini kit (for RNA) or QIAquick PCR Purification kit(for DNA) (QIAGEN Inc.—USA, Valencia, Calif.). For the QIAGEN columns,samples are applied twice. For washes, 80% EtOH is preferablysubstituted for the buffer provided with the QIAGEN kit. Samples areeluted twice with 50 μl volumes of 70° C. H₂O. Alternatively (but lesspreferably), samples may be cleaned up by repeated cycles of dilutionand concentration on Microcon-30 filters.

[0085] In a second step of the embodiment, α-nucleic acids arederivatized with NHS-esters, preferably Cy 3 or Cy 5. Preferably, 2-6 μgof aa-labeled nucleic acid are aliquoted into a microfuge tube,adjusting the total volume to 12 μl with H₂O. The NHS-ester is dissolvedat a concentration of ˜15 mM in anhydrous DMSO (˜200 nmoles in 13 μl).27 μl of 0.1 M sodium carbonate buffer, pH 9, are added. 12 μl of thedye mix (containing ˜60 nmoles dye-NHS ester) are then immediately addedto the aa-labeled nucleic acid (˜6-20 pmoles of a 1 kb molecule). Thesamples are then incubated in the dark at 23° C. for 1 hour. Thecoupling reaction is stopped by adding 5 μl of a 4M solution ofhydroxylamine. Incubation is continued at 23° C. for an additional 0.25hr. Dye-coupled nucleic acid is separated from unincorporated dye on anRNeasy® Mini kit or QIAquick PCR Purification kit (QIAGEN Inc.—USA,Valencia, Calif.). Samples are washed with 80% EtOH instead of buffer,as described above, and eluted twice with 50 μl volumes of 70° C. H₂O.

[0086] The spectrum of the labeled nucleic acid is preferably measuredfrom 220 nm-700 nm. The percent recovery of nucleic acid and molarincorporation of dye is calculated from extinction coefficients andabsorbance values at 1_(max). Recovery of nucleic acid is typically˜80%. The mole percent of dye incorporated per nucleotide ranges from1.5-5% of total nucleotides.

[0087] Often it is desired to compare gene expression in two differentpopulations of cells, perhaps derived from different tissues or perhapsexposed to different stimuli. Such comparisons are facilitated bylabeling the RNAs from one population with a first fluorophore and theRNAs from the other population, with a second fluorophore, where the twofluorophores have distinct emission spectra. Again, Cy3 and Cy5 areparticularly preferred fluorophores for use in comparing gene expressionbetween two different populations of cells.

[0088] 5.5. Methods of Preparation of Source RNA

[0089] The source RNA may be obtained from a variety of differentsources, typically a biological source. In specific embodiments, thebiological source may be any of a variety of eukaryotic sources.Biological sources of interest may include sources derived fromsingle-celled organisms such as yeast and multicellular organisms,including plants and animals, particularly mammals. Biological sourcesfrom multicellular organisms may be derived from particular organs ortissues of the multicellular organism, or from isolated cells derivedtherefrom. In obtaining the sample of RNA to be analyzed from itsbiological source, the source may be subjected to a number of differentprocessing steps. Such processing steps might include tissuehomogenization, cell isolation followed by cytoplasm extraction orisolation, nucleic acid extraction and the like. Such processing stepsfor isolating RNA from its biological source are known to those of skillin the art. For example, methods of isolating RNA from cells, tissues,organs or whole organisms are described in Sambrook et al. (1989),Molecular Cloning: A Laboratory Manual 2d Ed. (Cold Spring HarborPress), incorporated herein by reference in its entirety. Alternatively,at least some of the initial steps of the subject methods may beperformed in situ, as described in Eberwine (U.S. Pat. No. 5,514,545,entitled “Method for characterizing single cells based on RNAamplification for diagnostics and therapeutics,” issued May 7, 1996),the disclosure of which is herein incorporated by reference.

[0090] Although the amplification methods of the invention can beadapted to amplify DNA, it is preferred to utilize the methods toamplify RNA from a population of cells. Total cellular RNA, cytoplasmicRNA, or poly(A)⁺ RNA may be used, with poly(A)⁺ RNA (mRNA) beingpreferred. Methods for preparing total and poly(A)⁺ RNA are well knownand are described generally in Sambrook et al. (1989, MolecularCloning—A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) and Ausubel et al., eds. (1994,Current Protocols in Molecular Biology, vol. 2, Current ProtocolsPublishing, New York), incorporated herein by reference in theirentireties.

[0091] RNA may be isolated from eukaryotic cells by procedures thatinvolve lysis of the cells and denaturation of the proteins containedtherein. Cells of interest include wild-type cells, drug-exposedwild-type cells, modified cells, and drug-exposed modified cells.

[0092] Additional steps may be employed to remove DNA. Cell lysis may beaccomplished with a nonionic detergent, followed by microcentrifugationto remove the nuclei and hence the bulk of the cellular DNA. In oneembodiment, RNA is extracted from cells of the various types of interestusing guanidinium thiocyanate lysis followed by CsCl centrifugation toseparate the RNA from DNA (Chirgwin et al., 1979, Biochemistry18:5294-5299). Poly(A)⁺ RNA is selected by selection with oligo-dTcellulose (see Sambrook et al., 1989, Molecular Cloning—A LaboratoryManual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). Alternatively, separation of RNA from DNA can beaccomplished by organic extraction, for example, with hot phenol orphenol/chloroform/isoamyl alcohol.

[0093] If desired, RNase inhibitors may be added to the lysis buffer.Likewise, for certain cell types, it may be desirable to add a proteindenaturation/digestion step to the protocol.

[0094] For many applications, it is desirable to preferentially enrichmRNA with respect to other cellular RNAs, such as transfer RNA (tRNA)and ribosomal RNA (rRNA). Most mRNAs contain a poly(A) tail at their 3′end. This allows them to be enriched by affinity chromatography, forexample, using oligo(dT) or poly(U) coupled to a solid support, such ascellulose or Sephadex™ (see Ausubel et al., eds., 1994, CurrentProtocols in Molecular Biology, vol. 2, Current Protocols Publishing,New York). Once bound, poly(A)⁺ mRNA is eluted from the affinity columnusing 2 mM EDTA/0.1% SDS.

[0095] The sample of RNA can comprise a plurality of different mRNAmolecules, each different mRNA molecule having a different nucleotidesequence. In a specific embodiment, the mRNA molecules in the RNA samplecomprise at least 100 different nucleotide sequences. More preferably,the mRNA molecules of the RNA sample comprise at least 500, 1,000,5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,00090,000 or 100,000 different nucleotide sequences. In another specificembodiment, the RNA sample is a mammalian RNA sample, the mRNA moleculesof the mammalian RNA sample comprising about 20,000 to 30,000 differentnucleotide sequences.

[0096] In a specific embodiment, total RNA or mRNA from cells are usedin the methods of the invention. The source of the RNA can be cells of aplant or animal, human, mammal, primate, non-human animal, dog, cat,mouse, rat, rabbit, bird, yeast, eukaryote, prokaryote, etc. In oneembodiment, the method of the invention is used with a sample containingtotal mRNA or total RNA from 1×10⁶ cells or less.

[0097] 5.6. Methods for Determining Biological Response Profiles

[0098] This section provides some exemplary methods for measuringbiological responses using cRNA amplified by methods of the invention.One of skill in the art would appreciate that this invention is notlimited to the following specific methods for measuring the responses ofa biological system, i.e., gene expression profiles. In particular, thepresence of cRNA(s) of interest (and thus mRNA(s) of interest in thesample) can be detected or measured by procedures including, but notlimited to, Northern blotting, the use of oligonucleotides tethered tobeads as probes, or the use of polynucleotide microarrays.

[0099] In a specific embodiment of the invention, one or more labels isintroduced into the RNA during the transcription step to facilitate geneexpression profiling. Gene expression can be profiled in any of severalways, among which the preferred method is to probe a DNA microarray withthe labeled RNA transcripts generated above. A DNA microarray, or chip,is a microscopic array of DNA fragments or synthetic oligonucleotides,disposed in a defined pattern on a solid support, wherein they areamenable to analysis by standard hybridization methods (Schena,BioEssays 18: 427, 1996).

[0100] The DNA in a microarray may be derived from genomic or cDNAlibraries, from fully sequenced clones, or from partially sequencedcDNAs known as expressed sequence tags (ESTs). Methods for obtainingsuch DNA molecules are generally known in the art (see, e.g., Ausubel etal., eds., 1994, Current. Protocols in Molecular Biology, vol. 2,Current Protocols Publishing, New York). Alternatively, oligonucleotidesmay be synthesized by conventional methods, such asphosphoramidite-based synthesis.

[0101] Gene expression profiling can be done for purposes of screening,diagnosis, staging a disease, and monitoring response to therapy, aswell as for identifying genetic targets of drugs and of pathogens.

[0102] 5.6.1. Transcript Assay Using DNA Arrays

[0103] This invention is particularly useful for the analysis of geneexpression profiles. For expression profiling, DNA microarrays aretypically probed using mRNA, extracted and amplified from the cellswhose gene expression profile it is desired to analyze, using therandom-primed RT-IVT amplification method of the invention. Tofacilitate comparison between any two samples of interest, thepolynucleotides representing the mRNA transcripts present in a cell aretypically labeled separately with fluorescent dyes that emit atdifferent wavelengths. Some embodiments of this invention are based onmeasuring the transcriptional rate of genes.

[0104] The transcriptional rate can be measured by techniques ofhybridization to arrays of nucleic acid or nucleic acid mimic probes,described in the next subsection, or by other gene expressiontechnologies, such as those described in the subsequent subsection.However measured, the result is either the absolute, relative amounts oftranscripts or response data including values representing RNA abundanceratios, which usually reflect DNA expression ratios (in the absence ofdifferences in RNA degradation rates).

[0105] In various alternative embodiments of the present invention,aspects of the biological state other than the transcriptional state,such as the translational state, the activity state, or mixed aspectscan be measured.

[0106] Preferably, measurement of the transcriptional state is made byhybridization to transcript arrays, which are described in thissubsection. Certain other methods of transcriptional state measurementare described later in this subsection.

[0107] In a preferred embodiment the present invention makes use of“transcript arrays” (also called herein “microarrays”). Transcriptarrays can be employed for analyzing the transcriptional state in abiological sample and especially for measuring the transcriptionalstates of a biological sample exposed to graded levels of a drug ofinterest or to graded perturbations to a biological pathway of interest.

[0108] In one embodiment, transcript arrays are produced by hybridizingdetectably labeled polynucleotides representing the mRNA transcriptspresent in a cell (e.g., fluorescently labeled cRNA that is amplified bythe methods of the present invention) to a microarray. A microarray is asurface with an ordered array of binding (e.g., hybridization) sites forproducts of many of the genes in the genome of a cell or organism,preferably most or almost all of the genes. Microarrays can be made in anumber of ways, of which several are described below. However produced,microarrays share certain preferred characteristics: The arrays arereproducible, allowing multiple copies of a given array to be producedand easily compared with each other. Preferably the microarrays aresmall, usually smaller than 5 cm², and they are made from materials thatare stable under binding (e.g., nucleic acid hybridization) conditions.A given binding site or unique set of binding sites in the microarraywill specifically bind the product of a single gene in the cell.Although there may be more than one physical binding site (hereinafter“site”) per specific mRNA for the sake of clarity the discussion belowwill assume that there is a single site.

[0109] In one embodiment, the microarray is an array of polynucleotideprobes, the array comprising a support with at least one surface and atleast 100 different polynucleotide probes, each different polynucleotideprobe comprising a different nucleotide sequence and being attached tothe surface of the support in a different location on the surface.Preferably, the nucleotide sequence of each of the differentpolynucleotide probes is in the range of 40 to 80 nucleotides in length.More preferably, the nucleotide sequence of each of the differentpolynucleotide probes is in the range of 50 to 70 nucleotides in length.Even more preferably, the nucleotide sequence of each of the differentpolynucleotide probes is in the range of 50 to 60 nucleotides in length.

[0110] In specific embodiments, the array comprises polynucleotideprobes of at least 2,000, 4,000, 10,000, 15,000, 20,000, 50,000, 80,000,or 100,000 different nucleotide sequences.

[0111] In another embodiment, the nucleotide sequence of eachpolynucleotide probe in the array is specific for a particular targetpolynucleotide sequence. In yet another embodiment, the targetpolynucleotide sequences comprise expressed polynucleotide sequences ofa cell or organism.

[0112] In a specific embodiment, the cell or organism is a mammaliancell or organism. In another specific embodiment, the cell or organismis a human cell or organism.

[0113] In specific embodiments, the nucleotide sequences of thedifferent polynucleotide probes of the array are specific for at least50%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99% of the genes in the genome of the cell or organism.Most preferably, the nucleotide sequences of the differentpolynucleotide probes of the array are specific for all of the genes inthe genome of the cell or organism.

[0114] In specific embodiments, the polynucleotide probes of the arrayhybridize specifically and distinguishably to at least 10,000, to atleast 20,000, to at least 50,000, different polynucleotide sequences, toat least 80,000, or to at least 100,000 different polynucleotidesequences.

[0115] In other specific embodiments, the polynucleotide probes of thearray hybridize specifically and distinguishably to at least 90%, atleast 95%, or at least 99% of the genes or gene transcripts of thegenome of a cell or organism. Most preferably, the polynucleotide probesof the array hybridize specifically and distinguishably to the genes orgene transcripts of the entire genome of a cell or organism.

[0116] In specific embodiments, the array has at least 100, at least250, at least 1,000, or at least 2,500 probes per 1 cm², preferably allor at least 25% or 50% of which are different from each other.

[0117] In another embodiment, the array is a positionally addressablearray (in that the sequence of the polynucleotide probe at each positionis known).

[0118] In another embodiment, the nucleotide sequence of eachpolynucleotide probe in the array is a DNA sequence. In anotherembodiment, the DNA sequence is a single-stranded DNA sequence. The DNAsequence may be, e.g., a cDNA sequence, or a synthetic sequence.

[0119] When cRNA complementary to the RNA of a cell is made andhybridized to a microarray under suitable hybridization conditions, thelevel of hybridization to the site in the array corresponding to anyparticular gene will reflect the prevalence in the cell of mRNAtranscribed from that gene. For example, when detectably labeled (e.g.,with a fluorophore) cRNA complementary to the total cellular mRNA ishybridized to a microarray, the site on the array corresponding to agene (i.e., capable of specifically binding the product of the gene)that is not transcribed in the cell will have little or no signal (e.g.,fluorescent signal), and a gene for which the encoded mRNA is prevalentwill have a relatively strong signal.

[0120] In preferred embodiments, cRNAs from two different cells arehybridized to the binding sites of the microarray. In the case of drugresponses one biological sample is exposed to a drug and anotherbiological sample of the same type is not exposed to the drug. In thecase of pathway responses one cell is exposed to a pathway perturbationand another cell of the same type is not exposed to the pathwayperturbation. The cRNA derived from each of the two cell types aredifferently labeled so that they can be distinguished. In oneembodiment, for example, cRNA from a cell treated with a drug (orexposed to a pathway perturbation) is synthesized using afluorescein-labeled NTP, and cRNA from a second cell, not drug-exposed,is synthesized using a rhodamine-labeled NTP. When the two cRNAs aremixed and hybridized to the microarray, the relative intensity of signalfrom each cRNA set is determined for each site on the array, and anyrelative difference in abundance of a particular mRNA detected.

[0121] In the example described above, the cRNA from the drug-treated(or pathway perturbed) cell will fluoresce green when the fluorophore isstimulated and the cRNA from the untreated cell will fluoresce red. As aresult, when the drug treatment has no effect, either directly orindirectly, on the relative abundance of a particular mRNA in a cell,the mRNA will be equally prevalent in both cells and, upon reversetranscription, red-labeled and green-labeled cRNA will be equallyprevalent. When hybridized to the microarray, the binding site(s) forthat species of RNA will emit wavelengths characteristic of bothfluorophores (and appear brown in combination). In contrast, when thedrug-exposed cell is treated with a drug that, directly or indirectly,increases the prevalence of the mRNA in the cell, the ratio of green tored fluorescence will increase. When the drug decreases the mRNAprevalence, the ratio will decrease.

[0122] The use of a two-color fluorescence labeling and detection schemeto define alterations in gene expression has been described, e.g., inSchena et al., 1995, Science 270:467-470, which is incorporated byreference in its entirety for all purposes. An advantage of using cRNAlabeled with two different fluorophores is that a direct and internallycontrolled comparison of the mRNA levels corresponding to each arrayedgene in two cell states can be made, and variations due to minordifferences in experimental conditions (e.g., hybridization conditions)will not affect subsequent analyses. However, it will be recognized thatit is also possible to use cRNA from a single cell, and compare, forexample, the absolute amount of a particular mRNA in, e.g., adrug-treated or pathway-perturbed cell and an untreated cell.

[0123] 5.6.2. Preparation of Microarrays

[0124] Microarrays are known in the art and consist of a surface towhich probes that correspond in sequence to gene products (e.g., cDNAs,mRNAs, cRNAs, polypeptides, and fragments thereof), can be specificallyhybridized or bound at a known position. In one embodiment, themicroarray is an array (i.e., a matrix) in which each positionrepresents a discrete binding site for a product encoded by a gene(e.g., a protein or RNA), and in which binding sites are present forproducts of most or almost all of the genes in the organism's genome. Ina preferred embodiment, the “binding site” (hereinafter, “site”) is anucleic acid or nucleic acid analogue to which a particular cognate cRNAcan specifically hybridize. The nucleic acid or analogue of the bindingsite can be, e.g., a synthetic oligomer, a full-length cRNA, a less-thanfull length cRNA, or a gene fragment.

[0125] In one embodiment, the microarray contains binding sites forproducts of all or almost all genes in the target organism's genome.This microarray will have binding sites corresponding to at least about50% of the genes in the genome, often at least about 75%, more often atleast about 85%, even more often more than about 90%, and most often atleast about 99%.

[0126] Such comprehensiveness, however, is not necessarily required. Inanother embodiment, the microarray contains binding sites for productsof human genes. This microarray will have binding sites corresponding toat least about 5-10% of the genes in the genome, preferably at leastabout 10-15%, and more preferably at least about 40%.

[0127] Preferably, the microarray has binding sites for genes relevantto the action of a drug of interest or in a biological pathway ofinterest. A “gene” is identified as an open reading frame (ORF) ofpreferably at least 50, 75, or 99 amino acids from which a messenger RNAis transcribed in the organism (e.g., if a single cell) or in some cellin a multicellular organism. The number of genes in a genome can beestimated from the number of mRNAs expressed by the organism, or byextrapolation from a well-characterized portion of the genome. When thegenome of the organism of interest has been sequenced, the number ofORFs can be determined and mRNA coding regions identified by analysis ofthe DNA sequence. For example, the Saccharomyces cerevisiae genome hasbeen completely sequenced and is reported to have approximately 6275open reading frames (ORFs) longer than 99 amino acids. Analysis of theseORFs indicates that there are 5885 ORFs that are likely to specifyprotein products (Goffeau et al., 1996, Science 274:546-567, which isincorporated by reference in its entirety for all purposes). Incontrast, the human genome is estimated to contain approximately 10⁵genes.

[0128] 5.6.3. Preparation of Nucleic Acids for Microarrays

[0129] As noted above, the “binding site” to which a particular cognatecRNA specifically hybridizes is usually a nucleic acid or nucleic acidanalogue attached at that binding site. In one embodiment, the bindingsites of the microarray are DNA polynucleotides corresponding to atleast a portion of each gene in an organism's genome. These DNAs can beobtained by, e.g., polymerase chain reaction (PCR) amplification of genesegments from genomic DNA, cDNA (e.g., by reverse transcription orRT-PCR), or cloned sequences. Nucleic acid amplification primers arechosen, based on the known sequence of the genes or cDNA, that result inamplification of unique fragments (i.e., fragments that do not sharemore than 10 bases of contiguous identical sequence with any otherfragment on the microarray). Computer programs are useful in the designof primers with the required specificity and optimal amplificationproperties. See, e.g., Oligo version 5.0 (National Biosciences). In thecase of binding sites corresponding to very long genes, it willsometimes be desirable to amplify segments near the 3′ end of the geneso that when oligo-dT primed cDNA probes are hybridized to themicroarray, less-than-full length probes will bind efficiently.Typically each gene fragment on the microarray will be between about 50bp and about 2000 bp, more typically between about 100 bp and about 1000bp, and usually between about 300 bp and about 800 bp in length.

[0130] Nucleic acid amplification methods are well known and aredescribed, for example, in Innis et al., eds., 1990, PCR Protocols: AGuide to Methods and Applications, Academic Press Inc., San Diego,Calif., which is incorporated by reference in its entirety for allpurposes. It will be apparent that computer controlled robotic systemsare useful for isolating and amplifying nucleic acids.

[0131] An alternative means for generating the nucleic acid for themicroarray is by synthesis of synthetic polynucleotides oroligonucleotides, e.g., using N-phosphonate or phosphoramiditechemistries (e.g., Froehler et al., 1986, Nucleic Acid Res14:5399-5407). Synthetic sequences are between about 15 and about 100bases in length, preferably between about 20 and about 50 bases.

[0132] In some embodiments, synthetic nucleic acids include non-naturalbases, e.g., inosine. Where the particular base in a given sequence isunknown or is polymorphic, a universal base, such as inosine or5-nitroindole, may be substituted. Additionally, it is possible to varythe charge on the phosphate backbone of the oligonucleotide, forexample, by thiolation or methylation, or even to use a peptide ratherthan a phosphate backbone. The making of such modifications is withinthe skill of one trained in the art.

[0133] As noted above, nucleic acid analogues may be used as bindingsites for hybridization. An example of a suitable nucleic acid analogueis peptide nucleic acid (see, e.g., Egholm et al., 1993, Nature365:566-568; see also U.S. Pat. No. 5,539,083, Cook et al., entitled“Peptide nucleic acid combinatorial libraries and improved methods ofsynthesis,” issued July 23, 1996).

[0134] In an alternative embodiment, the binding (hybridization) sitesare made from plasmid or phage clones of genes, cDNAs (e.g., expressedsequence tags), or inserts therefrom (Nguyen et al., 1995, Genomics29:207-209). In yet another embodiment, the polynucleotide of thebinding sites is RNA.

[0135] 5.6.4. Attaching Nucleic Acids to the Solid Surface

[0136] The nucleic acid or analogue are attached to a solid support,which may be made from glass, silicon, plastic (e.g., polypropylene,nylon, polyester), polyacrylamide, nitrocellulose, cellulose acetate orother materials. In general, non-porous supports, and glass inparticular, are preferred. The solid support may also be treated in sucha way as to enhance binding of oligonucleotides thereto, or to reducenon-specific binding of unwanted substances thereto. Preferably, theglass support is treated with polylysine or silane to facilitateattachment of oligonucleotides to the slide.

[0137] Methods of immobilizing DNA on the solid support may includedirect touch, micropipetting (Yershov et al., Proc. Natl. Acad. Sci. USA(1996) 93(10):4913-4918), or the use of controlled electric fields todirect a given oligonucleotide to a specific spot in the array (U.S.Pat. No. 5,605,662, Heller et al., entitled “Active programmableelectronic devices for molecular biological analysis and diagnostics,”issued Feb. 25, 1997). DNA is typically immobilized at a density of 100to 10,000 oligonucleotides per cm² and preferably at a density of about1000 oligonucleotides per cm².

[0138] A preferred method for attaching the nucleic acids to a surfaceis by printing on glass plates, as is described generally by Schena etal., 1995, Science 270:467-470. This method is especially useful forpreparing microarrays of cDNA. (See also DeRisi et al., 1996, NatureGenetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; andSchena et al., Proc. Natl. Acad. Sci. USA, 1996, 93(20):10614-19.)

[0139] In a preferred alternative to immobilizing pre-fabricatedoligonucleotides onto a solid support, it is possible to synthesizeoligonucleotides directly on the support (Maskos et al., Nucl. AcidsRes. 21: 2269-70, 1993; Fodor et al., Science 251: 767-73, 1991;Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20-4). Among methods ofsynthesizing oligonucleotides directly on a solid support, particularlypreferred methods are photolithography (see Fodor et al., Science 251:767-73, 1991; McGall et al., Proc. Natl. Acad. Sci. (USA) 93: 13555-60,1996) and piezoelectric printing (Lipshutz et al., 1999, Nat. Genet.21(1 Suppl):20-4), with the piezoelectric method most preferred.

[0140] In one embodiment, a high-density oligonucleotide array isemployed. Techniques are known for producing arrays containing thousandsof oligonucleotides complementary to defined sequences, at definedlocations on a surface using photolithographic techniques for synthesisin situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al.,1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; Lockhart et al., 1996,Nature Biotechnol. 14:1675-80; U.S. Pat. No. 5,578,832, Trulson et al.,entitled “Method and apparatus for imaging a sample on a device,” issuedNov. 26, 1996; U.S. Pat. No. 5,556,752, Lockhart et al., entitled“Surface-bound, unimolecular, double-stranded DNA,” issued Sep. 17,1996; and U.S. Pat. No. 5,510,270, Fodor et al., entitled “Synthesis andscreening of immobilized oligonucleotide arrays,” issued Apr. 23, 1996;each of which is incorporated by reference in its entirety for allpurposes) or other methods for rapid synthesis and deposition of definedoligonucleotides (Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20-4.)

[0141] When these methods are used, oligonucleotides (e.g., 20-mers) ofknown sequence are synthesized directly on a surface such as aderivatized glass slide. Usually, the array produced contains multipleprobes against each target transcript. Oligonucleotide probes can bechosen to detect alternatively spliced mRNAs or to serve as various typeof control.

[0142] In a particularly preferred embodiment, microarrays of theinvention are manufactured by means of an ink jet printing device foroligonucleotide synthesis, e.g., using the methods and systems describedby Blanchard in International Patent Publication No. WO 98/41531,published Sep. 24, 1998; Blanchard et al., 1996, Biosensors andBioeletronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arrays inGenetic Engineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New Yorkat pages 111-123; U.S. Pat. No. 6,028,189 to Blanchard. Specifically,the oligonucleotide probes in such microarrays are preferablysynthesized in arrays, e.g., on a glass slide, by serially depositingindividual nucleotide bases in “microdroplets” of a high surface tensionsolvent such as propylene carbonate. The microdroplets have smallvolumes (e.g., 100 pL or less, more preferably 50 pL or less) and areseparated from each other on the microarray (e.g., by hydrophobicdomains) to form circular surface tension wells which define thelocations of the array elements (i.e., the different probes).

[0143] Other methods for making microarrays, e.g., by masking (Maskosand Southern, 1992, Nuc. Acids Res. 20:1679-1684), may also be used. Inprincipal, any type of array, for example, dot blots on a nylonhybridization membrane (see Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.), could be used, although, as will berecognized by those of skill in the art, very small arrays will bepreferred because hybridization volumes will be smaller.

[0144] 5.6.5. Hybridization to Microarrays

[0145] Nucleic acid hybridization and wash conditions are optimallychosen so that the probe “specifically binds” or “specificallyhybridizes” to a specific array site, i.e., the probe hybridizes,duplexes or binds to a sequence array site with a complementary nucleicacid sequence but does not hybridize to a site with a non-complementarynucleic acid sequence. As used herein, one polynucleotide sequence isconsidered complementary to another when, if the shorter of thepolynucleotides is less than or equal to 25 bases, there are nomismatches using standard base-pairing rules or, if the shorter of thepolynucleotides is longer than 25 bases, there is no more than a 5%mismatch. Preferably, the polynucleotides are perfectly complementary(no mismatches). It can easily be demonstrated that specifichybridization conditions result in specific hybridization by carryingout a hybridization assay including negative controls (see, e.g. Shalonet al., 1996, Genome Research 6:639-645, and Chee et al., 1996, Science274:610-614).

[0146] Optimal hybridization conditions will depend on the length (e.g.,oligomer versus polynucleotide greater than 200 bases) and type (e.g.,RNA, DNA, PNA) of labeled probe and immobilized polynucleotide oroligonucleotide. General parameters for specific (i.e., stringent)hybridization conditions for nucleic acids are described in Sambrook etal. (1989, Molecular Cloning—A Laboratory Manual (2nd Ed.), Vols. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and in Ausubelet al. (1987, Current Protocols in Molecular Biology, Greene Publishing,Media, Pa., and Wiley-Interscience, New York). When the cDNA microarraysof Schena et al. (1996, Proc. Natl. Acad. Sci. USA, 93:10614-19) areused, typical hybridization conditions are hybridization in 5×SSC plus0.2% SDS at 65° C. for 4 hours followed by washes at 25° C. in lowstringency wash buffer (1×SSC plus 0.2% SDS) followed by 10 minutes at25° C. in high stringency wash buffer (0.1×SSC plus 0.2% SDS) (Schena etal., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-19). Usefulhybridization conditions are also provided in, e.g., Tijssen, 1993,Hybridization With Nucleic Acid Probes, Elsevier Science PublishersB.V., Amsterdam and New York, and Kricka, 1992, Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, Calif.

[0147] Although simultaneous hybridization of differentially labeledmRNA samples is preferred, it is also possible to use a single label andto perform hybridizations sequentially rather than simultaneously.

[0148] 5.6.6. Signal Detection and Data Analysis

[0149] When fluorescently labeled probes are used, the fluorescenceemissions at each site of a transcript array can be, preferably,detected by scanning confocal laser microscopy. In one embodiment, aseparate scan, using the appropriate excitation line, is carried out foreach of the two fluorophores used. Alternatively, a laser can be usedthat allows simultaneous specimen illumination at wavelengths specificto the two fluorophores and emissions from the two fluorophores can beanalyzed simultaneously (see Shalon et al., 1996, Genome Research6:639-645, which is incorporated by reference in its entirety for allpurposes). In a preferred embodiment, the arrays are scanned with alaser fluorescent scanner with a computer controlled X-Y stage and amicroscope objective. Sequential excitation of the two fluorophores isachieved with a multi-line, mixed gas laser and the emitted light issplit by wavelength and detected with two photomultiplier tubes.Fluorescence laser scanning devices are described in Shalon et al.,1996, Genome Res. 6:639-645 and in other references cited herein.Alternatively, the fiber-optic bundle described by Ferguson et al.,1996, Nature Biotechnol. 14:1681-1684, may be used to monitor mRNAabundance levels at a large number of sites simultaneously.

[0150] Signals are recorded and, in a preferred embodiment, analyzed bycomputer, e.g., using a 12 bit analog to digital board. In oneembodiment the scanned image is despeckled using a graphics program(e.g., Hijaak Graphics Suite) and then analyzed using an image griddingprogram that creates a spreadsheet of the average hybridization at eachwavelength at each site. If necessary, an experimentally determinedcorrection for “cross talk” (or overlap) between the channels for thetwo fluors may be made. For any particular hybridization site on thetranscript array, a ratio of the emission of the two fluorophores can becalculated. The ratio is independent of the absolute expression level ofthe cognate gene, but is useful for genes whose expression issignificantly modulated by drug administration, gene deletion, or anyother tested event.

[0151] According to the method of the invention, the relative abundanceof an mRNA in two biological samples is scored as a perturbation and itsmagnitude determined (i.e., the abundance is different in the twosources of mRNA tested), or as not perturbed (i.e., the relativeabundance is the same). In various embodiments, a difference between thetwo sources of RNA of at least a factor of about 25% (RNA from onesource is 25% more abundant in one source than the other source), moreusually about 50%, even more often by a factor of about 2 (twice asabundant), 3 (three times as abundant) or 5 (five times as abundant) isscored as a perturbation.

[0152] Preferably, in addition to identifying a perturbation as positiveor negative, it is advantageous to determine the magnitude of theperturbation. This can be carried out, as noted above, by calculatingthe ratio of the emission of the two fluorophores used for differentiallabeling, or by analogous methods that will be readily apparent to thoseof skill in the art.

[0153] In one embodiment, two samples, each labeled with a differentfluor, are hybridized simultaneously to permit differential expressionmeasurements. If neither sample hybridizes to a given spot in the array,no fluorescence will be seen. If only one hybridizes to a given spot,the color of the resulting fluorescence will correspond to that of thefluor used to label the hybridizing sample (for example, green if thesample was labeled with Cy3, or red, if the sample was labeled withCy5). If both samples hybridize to the same spot, an intermediate coloris produced (for example, yellow if the samples were labeled withfluorescein and rhodamine). Then, applying methods of patternrecognition and data analysis known in the art, it is possible toquantify differences in gene expression between the samples. Methods ofpattern recognition and data analysis are described in e.g., co-pendingU.S. patent application Ser. No. 09/179,569 filed on Oct. 27, 1998, byFriend et al.; Ser. No. 09/220,142 filed on Dec. 23, 1998, by Stoughtonet al.; Ser. No. 09/220,275 filed on Dec. 23, 1998, by Friend et al.;International Publication WO 00/24936, dated May 4, 2000, which areincorporated by reference herein in their entireties.

[0154] 5.7. Diagnostic Methods

[0155] The random-primed RT-IVT methods of the invention have use innucleic acid amplification reactions to generate sufficient quantitiesof nucleic acid for detection of a specific nucleic acid of interest.Accordingly, the methods of the invention can be used in methods ofdiagnosis, for example, in amplifying a sequence (e.g., genomic) of aninfectious disease agent, e.g., of human disease including but notlimited to viruses, bacteria, parasites, and fungi, thereby diagnosingthe presence of the infectious agent in a sample of nucleic acid from apatient. The nucleic acid of interest can be genomic or cDNA or mRNA, orcan be synthetic, human or animal, or of a microorganism, etc. Inanother embodiment that can be used in the diagnosis or prognosis of adisease or disorder, the nucleic acid of interest is a wild type humangenomic or RNA or cDNA sequence, mutation of which is implicated in thepresence of a human disease or disorder, or alternatively, can be themutated sequence. By way of example, the mutation can be an insertion,substitution, and/or deletion of one or more nucleotides, or atranslocation.

[0156] 5.8. Kits for the Amplification and Detection of Selected TargetNucleotide Sequences

[0157] The present invention also provides kits for the linearamplification of RNA, and, for example, detection or measurement ofnucleic acid amplification products and for determining the responses orstate of a biological sample. Such a kit may comprise containers, eachwith one or more of the various reagents (typically in concentratedform) utilized in the methods of the invention, including, for example,buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP,dGTP, dTTP, ATP, CTP, GTP and UTP), reverse transcriptase, RNApolymerase specific to the RNA polymerase promoter, and the randompromoter-primers and primers of the present invention. Optionally alsopresent in the kit is a reverse transcriptase inhibitor, where, in manyembodiments, the inhibitor is at least ddNTP or a combination of ddNTPs,e.g., ddATP and/or ddGTP. A set of instructions for use of kitcomponents in an mRNA amplification method of the present invention,will also be typically included.

[0158] In a specific embodiment, the kit comprises one or more primeroligonucleotides of the invention, such as a RNA polymerasepromoter-containing primer, including but not limited to a set of randomRNA polymerase promoter-containing primers and/or a set of randomprimers, in one or more containers. The kit can comprise for example, arandom T7-poly dN primer set, a T7-poly dT primer, and/or a random polydN primer set. The kit can further comprise additional components forcarrying out the amplification reactions of the invention, such asreverse transcriptase and RNA polymerase. Where the target nucleic acidsequence being amplified is one implicated in disease or disorder, thekit can be used for diagnosis or prognosis.

[0159] Oligonucleotides in containers can be in any form, e.g.,lyophilized, or in solution (e.g., a distilled water or bufferedsolution), etc. Oligonucleotides ready for use in the same amplificationreaction can be combined in a single container or can be in separatecontainers.

[0160] The kit optionally further comprises a control nucleic acid,and/or a microarray, and/or means for stimulating and detectingfluorescent light emissions from fluorescently labeled RNA, and/orexpression profile projection and analysis software capable of beingloaded into the memory of a computer system. The kit optionally furtherprovides means for stimulating and detecting fluorescent lightemissions, e.g., a fluorescence plate reader or a combinationthermocycler-plate-reader to perform the analysis.

[0161] 5.8.1. Analytic Kit Implementation

[0162] In a preferred embodiment, the methods of this invention can beimplemented by use of kits containing oligonucleotide primers of theinvention and microarrays. The microarrays contained in such kitscomprise a solid phase, e.g., a surface, to which probes are hybridizedor bound at a known location of the solid phase. Preferably, theseprobes consist of nucleic acids of known, different sequence, with eachnucleic acid being capable of hybridizing to a RNA species or to a cDNAspecies derived therefrom. In particular, the probes contained in thekits of this invention are nucleic acids capable of hybridizingspecifically to nucleic acid sequences derived from RNA species that areknown to increase or decrease in response to perturbations to theparticular protein whose activity is determined by the kit. The probescontained in the kits of this invention preferably substantially excludenucleic acids that hybridize to RNA species that are not increased inresponse to perturbations to the particular protein whose activity isdetermined by the kit.

[0163] In another preferred embodiment, a kit of the invention furthercontains expression profile projection and analysis software capable ofbeing loaded into the memory of a computer system. An example of such asystem is described in co-pending U.S. patent application Ser. No.09/220,276, by Bassett, Jr. et al., filed Dec. 23, 1998, which isincorporated herein by reference in its entirety. Preferably, theexpression profile analysis software contained in a kit of thisinvention, is essentially identical to the expression profile analysissoftware 512 described in U.S. patent application Ser. No. 09/220,276.

[0164] Alternative kits for implementing the analytic methods of thisinvention will be apparent to one of skill in the art and are intendedto be comprehended within the accompanying claims. In particular, theaccompanying claims are intended to include the alternative programstructures for implementing the methods of this invention that will bereadily apparent to one of skill in the art.

[0165] The following experimental examples are offered by way ofillustration and not by way of limitation.

6. EXAMPLE 1 cDNA Synthesis and RNA Amplification for the Preparation ofCY3- and CY5-Labeled RNA Targets for Gene Expression Monitoring

[0166] This example demonstrates that using the random-primed RT-IVTmethod of the invention, linear amplification of mRNA to can be used toproduce unbiased antisense RNA profiles. The results of an mRNAamplification produced using the random-primed RT-IVT method of theinvention were compared with results obtained using the mRNAamplification method disclosed in Shannon (U.S. Pat. No. 6,132,997,entitled “Method for linear mRNA amplification,” issued Oct. 17, 2000).Using the random-primed RT-IVT method, poly-A⁺ RNA was converted todouble-stranded cDNA using degenerate random primers comprising a T7 RNApolymerase promoter sequence (T7-dN₉) to prime first strand cDNAsynthesis and degenerate random primers (dN₆) to prime second strandcDNA synthesis to yield a double-stranded cDNA that is recognized by T7RNA polymerase. The double-stranded cDNA was then transcribed intoantisense RNA by T7 RNA polymerase in the presence of a reversetranscriptase that was rendered incapable of RNA-dependent DNApolymerase activity during this transcription step by heat inactivation.5-(3-Aminoallyl)uridine 5′-triphosphate was incorporated into theantisense RNA during transcription and post-synthetically labeled withCy3-NHS or Cy5-NHS. Linear amplification extents of at least 100-foldand labeling efficiencies of approximately 3% were achieved using thismethod.

[0167] 6.1. Materials and Methods

[0168] Total RNA was isolated from Jurkat and K562 cell lines. Poly-A⁺RNA was isolated from the total RNA to provide the initial source mRNAused in the experiment.

[0169] cDNA Synthesis Reagents:

[0170] 1. mRNA, 0.2 mg.

[0171] 2. DNA T7-dN₉ (20 μM): (5′) AAT TAA TAC GAC TCA CTA TAG GGA GATNNN NNN NNN (3′) (N=A, T, C or G) (SEQ ID NO.: 1)

[0172] 3. MMLV Reverse Transcriptase (50 U/μl), Epicentre P/N M4425H

[0173] 4. RNAGuard™, Pharmacia P/N 27-0815-01

[0174] 5. 5× First Strand Buffer: 250 mM Tris-HCl, pH 8.3, 15 mM MgCl₂,375 mM KCl, Life Technologies P/N 18057-018

[0175] 6. 100 mM DTT* (*supplied with MMLV Reverse Transcriptase,Epicentre)

[0176] 7. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01

[0177] 8. ultraPURE distilled water, DNAse, RNAse Free, LifeTechnologies, Cat # 10977-015

[0178] 9. pdN₆ (200 ng/μl), diluted from Amersham Pharmacia Biotech P/N27-2166-01

[0179] Transcription Reagents:

[0180] 1. T7 RNA Polymerase (2500 units/μl), Epicentre P/N TU950K

[0181] 2. RNAGuard™, Pharmacia P/N 27-0815-01

[0182] 3. Inorganic Pyrophosphatase (200 U/ml), New England Biolabs,#M0296S.

[0183] 4. 5× Transcription Buffer: 0.2 M Tris-HCl, pH 7.5, 50 mM NaCl,30 mM MgCl₂, 10 mM spermidine, Epicentre PIN BP1001

[0184] 5. 100 mM DTT, Epicentre PIN BP1100

[0185] 6. MgCl₂ (200 mM), diluted from Sigma P/N M-1028

[0186] 7. NTPs (25 mM ATP, GTP, CTP, 6 mM UTP), diluted from PharmaciaP/N 27-2025-01

[0187] 8. 5-(3-Aminoallyl)uridine 5′-triphosphate (75 mM), Sigma P/NA-5660

[0188] 9. ultraPURE distilled water, DNAse, RNAse Free, LifeTechnologies, P/N 10977-015

[0189] Purification and Labeling Reagents:

[0190] 1. RNeasy® Mini Kit (250), QIAGEN Inc., P/N 74106

[0191] 2. Carbonate-Bicarbonate Buffer capsules, Sigma, PIN C-3041

[0192] 3. Hydrochloric acid, Fisher, P/N A508-500

[0193] 4. Anhydrous MSO (methyl sulfoxide, also known as DMSO, dimethylsulfoxide), Aldrich, PIN 27,685-5

[0194] 5. Cy3-NHS dye pack, Amersham, P/N PA23001

[0195] 6. Cy5-NHS dye pack, Amersham, P/N PA25001

[0196] 7. Hydroxylamine (“HA”), Sigma, P/N H-2391

[0197] Other materials:

[0198] 1. Pipetman micropipettors, (P-10, P-20, P-200, P-1000), orequivalent

[0199] 2. Sterile, nuclease-free 1.5 ml microcentrifuge tubes

[0200] 3. Sterile, nuclease-free aerosol-barrier pipet tips

[0201] 4. Thermal Cycler

[0202] Reagent Preparation:

[0203] 1. dNTPs (10 mM each)

[0204] Thaw dNTP stocks (100 mM) and place on ice. Add 10 μl each dNTPto 60 μl nuclease-free water. Store frozen.

[0205] 2. pdN₆ (200 ng/μl)

[0206] Add 663 μl nuclease-free water to lyophilized sample (50 A260units or approximately 1325 μg) for 2.0 μg/μl. Add 10 μl pdN₆ (2.0μg/μl) to 90 μl nuclease-free water for 200 ng/μl. Store frozen.

[0207] 3. 200 mM MgCl₂

[0208] Add 100 μl of 1 M MgCl₂ to 400 μl nuclease-free water. Storefrozen.

[0209] 4. NTPs (25 mM ATP, GTP, CTP, 6.0 mM UTP)

[0210] Thaw NTP stocks (100 mM) and place on ice. Combine 125 μl ATP,125 μl GTP, 125 μl CTP, 30 μl UTP and 95 μl nuclease-free water. Storefrozen.

[0211] 5. aa UTP (75 mM) Dissolve 5 mg in 125 μl water.

[0212] 6. Anhydrous MSO should be stored with a molecular sieve toabsorb water.

[0213] Procedure:

[0214] To prevent contamination of reactions by ribonucleases,laboratory gloves were worn and dedicated solutions and pipettors withnuclease-free, aerosol-resistant tips were used.

[0215] Amplified RNA preparations were prepared in batches of no lessthan 6 to minimize errors associated with pipetting small volumes ofenzyme solutions. The procedure below specifies reagent volumes for 1reaction; for 6 reactions, the specified volumes were multiplied by 6.5.

[0216] 1. Add 0.2 μg of source mRNA to reaction tube. Add 1.0 μl DNAT7-dN₉ (20 μM) and bring total sample volume to 10.5 μl in nuclease-freewater.

[0217] 2. Incubate at 65° C. for 10 min to denature primer and template.Move reaction tubes to ice. Store reactions tubes on ice for 5 min.

[0218] 3. Mix the following components and maintain on ice. cDNA MixComponent Volume (μl) 5x First Strand Buffer 4.0 100 mM DTT 2.0 dNTPs(10 mM each) 1.0 pdN₆ (200 ng/μl) 1.0 MMLV-RT (50 U/μl) 1.0 RNAGuard ™(36 U/μl) 0.5 Volume of cDNA Mix 9.5

[0219] 4. Aliquot 9.5 μl of cDNA Mix into each sample tube. IncubatecDNA synthesis reaction at 40° C. for 120 min. Composition of cDNASynthesis Reaction Final concentration Component or amount poly-A⁺ RNA200 ng DNA T7T18VN 1 μM Tris-HCl, pH 8.3 50 mM MgCl₂ 3.0 mM KCl 75 mMDTT 10 mM dNTPs 0.5 mM each MMLV-RT 50 U RNAGuard ™ 18 U Total reactionvolume 20 μl

[0220]  Incubate reaction tubes at 65° C. for 15 min. This inactivatesthe reverse transcriptase activity of MMLV prior to the IVT step. Movereaction tubes to ice. Store reaction tubes on ice for 5 min.

[0221] 5. Immediately before use, mix the following components in theorder indicated at room temperature: Transcription Mix Component Volume(μl) Nuclease-free water 22.8 5x Transcription Buffer 16 100 mM DTT 6.0NTPs (25 mM A, G, C, 6.0 mM UTP) 8.0 aa UTP (75 mM) 2.0 200 mM MgCl₂ 3.3RNAGuard ™ (36 U/μl) 0.5 Inorganic Pyrophosphatase (200 U/ml) 0.6 T7 RNApolymerase (2500 U/μl) 0.8 Volume of Transcription Mix 60

[0222] 6. Aliquot 60 μl of Transcription Mix into each sample tube.Incubate transcription reactions at 40° C. for 16 hrs. Composition ofTranscription Reaction Final concentration Component or amountDouble-strand cDNA Approximately 400 ng Tris-HCl, pH 7.5 52 mM MgCl₂ 15mM KCl 19 mM NaCl 10 mM Spermidine 2 mM DTT 10 mM ATP, GTP, CTP 2.5 mMeach UTP 0.6 mM aa UTP 1.9 mM T7 RNA polymerase 2000 U RNAGuard ™ 18 UInorganic pyrophosphatase 0.12 U Total reaction volume 80 μl

[0223] 7. RNeasy® (QIAGEN Inc.) Purification of reactions:

[0224] Add 20 μl water to 80 μl reaction tube.

[0225] Transfer to mixing tube.

[0226] Add 350 μl RLT buffer (QIAGEN Inc.) (plus 2-β-mercaptoethanol),mix well.

[0227] Add 250 μl 100% EtOH, mix well.

[0228] Transfer to RNeasy® column.

[0229] Spin 30 seconds in microfuge, 10 K.

[0230] Transfer column to new collection tube.

[0231] Add 7001 μl 80% EtOH.

[0232] Spin 30 seconds in microfuge, 10 K.

[0233] Discard flow through.

[0234] Add 700 μl 80% EtOH.

[0235] Spin 30 seconds in microfuge, 10 K.

[0236] Transfer column to new collection tube.

[0237] Spin 2 minutes, 14 K to dry filter.

[0238] Place column in microfuge tube.

[0239] Add 55 μl of nuclease-free water to filter. Let sit 1 minute.

[0240] Spin 14 K, 2 minutes.

[0241] Add 55 μl of nuclease-free water to filter. Let sit 1 minute.

[0242] Spin 14 K, 2 minutes.

[0243] To quantitate the yield of amplified RNA product, remove a 10.0μl aliquot of the product and dilute into 90 μl dH₂O. Add samples to aCostar UV-transparent plate and measure A260, A280 using a Spectramax(GRM Reader) and template for whichever lot of plates you are using.Calculate yield using the relationship A260=1 corresponds to 40 μg/ml.Conversion factor for Spectramax=3.59 (i.e. multiply A260 by 3.59 whencalculating yield).

[0244] 8. In speed vac, dry down 10 μg per fluor-reversed pair.

[0245] 9. Coupling Reactions:

[0246] Resuspend 10 μg IVT product in 7 μl water (or water plus E1a) anddivide into two tubes. One tube will be coupled with Cy3 and one withCy5.

[0247] Preparation of 3× Sodium Bicarbonate Buffer:

[0248] Place the contents of one Carbonate-Bicarbonate Buffer capsule(Sigma, P/N C-3041) into a 50 ml Falcon tube.

[0249] Add 16.7 ml RNase free water and mix well.

[0250] Add 125 μl 37% HCl and mix.

[0251] pH should be 9-9.5.

[0252] Preparation of Cy-NHS Dyes:

[0253] Spin dye briefly before opening tube.

[0254] Add 10 μl anhydrous MSO to dye.

[0255] Mix by pipetting 20 times.

[0256] Set a pipettman at 3.5 μl.

[0257] Work quickly since the amino esters are unstable in aqueousenvironment.

[0258] Add 20 μl 3× sodium bicarbonate buffer to dye and mix well.

[0259] Add 3.5 μl dye to each tube of cRNA. Mix well.

[0260] Incubate in the dark for 1 hour.

[0261] Stop the reaction by adding 3.5 μl 4M HA (hydroxylamine)

[0262] Incubate 10 minutes.

[0263] 10. Repeat RNeasy® clean-up as in Step 7, above, except elute in70° C. nuclease-free water.

[0264] 11. Measure yield and percent incorporation in a Costar UV plate.Calculate concentration of RNA using 1 OD₂₆₀=40 μg/ml RNA. Overallamplification yield is calculated by multiplying RNA concentration(μg/ml) by the sample volume (0.1 ml) and dividing by the amount ofpoly-A⁺ RNA initially added to the reaction. Calculate concentration ofCy3-CTP using ε(552 nm)=150 (1/mMcm). Calculate concentration of Cy5-CTPusing ε(650 run)=250 (1/mMcm).

[0265] Generation of Gene Expression Profile Signatures:

[0266] Source mRNA from Jurkat and K562 cell lines was used to generategene expression profile signatures by amplification and labeling usingthe Shannon method and using the random-primed RT-IVT method, followedby hybridization to DNA microarrays. Approximately 5 μg of Cy-labeledcRNA from each cell line was hybridized as fluor-reversed pairs to a DNAmicroarray pattern with probes tiled (overlapped) across all mRNAsequence for approximately 33 RefSeq test genes (LocusLink database,www.ncbi.nlm.nih.gov/locuslink/build.html) known to exhibit 3′amplification bias when amplified by the Shannon method. Analysis wasperformed either on a gene-by-gene basis or with all oligonucleotides atonce. The first goal of the study was to determine whether therandom-primed RT-IVT method produced a full-length cRNA. The second goalof the study was to determine whether the random-primed RT-IVT methodhas less of a 3′ bias when compared with the Shannon method.

[0267]6.2. Results and Discussion

[0268]FIG. 1 compares the profiles obtained from single-gene analysisusing the mRNA amplification method described in U.S. Pat. No. 6,132,997(Shannon, issued Oct. 17, 2000) (“Shannon”) and the random-primed RT-IVTmethod of the invention. The graphs plot signal intensity (mlavg) ofoligonucleotides in a single gene (X-axis) as a function of the numberof bp from the 5′ end (Y-axis). The 3′ bias of signal intensity seenwhen the Shannon method is used cannot be seen when the random-primedRT-IVT method is used, indicating that the random-primed RT-IVT methodovercomes the 3′ bias of the Shannon method.

[0269]FIG. 2 shows the intensity difference as a function of distancefrom the 3′ end. The graph shows the intensity of all oligonucleotidesas a function of distance from the 3′ end. The graph plots mlavg(Shannon method)—mlavg (random-primed RT-IVT method) (X-axis) versuslog₁₀ of the number of bp from the 3′ end (Y-axis). The intensityobtained with the Shannon method is greater than the intensity obtainedwith the random-primed RT-IVT method for probes less than 1000 bp fromthe 3′ end of the message. The intensity obtained with the Shannonmethod is less than the intensity obtained with the random-primed RT-IVTmethod for probes greater than 1000 bp from the 3′ end of the message.

[0270] At xdev threshold 2.5 (˜Pvalue 1%), the following number ofsignatures were obtained (Table 2): TABLE 2 Forward Reverse total bp <1000 bp > 1000 total bp < 1000 bp > 1000 random- 2486 1488 998 2237 1367870 primed RT-IVT Shannon 2587 1927 660 1218 892 326 method # of 74162965 4451 7413 2962 4451 probes

[0271] FIGS. 3(A-C) shows the signature differences in the numbers andpercentages of significant data points. The top graph (A) plots thenumber of probes (X-axis) versus the [log₁₀] (bp) (Y-axis). The middlegraph (B) plots the number of signatures (X-axis) versus the [log₁₀](bp) (Y-axis). The bottom graph (C) plots the fraction of signaturesversus the [log₁₀] (bp) (Y-axis). As can be seen in the bottom graph,the random-primed RT-IVT method outcompetes the Shannon method forprobes greater than 1000 bp from the 3′ end. Note the black arrow atapproximately 700 bp where random-primed RT-IVT method becomes betterthan the Shannon method. Stars: Shannon method. Circles: random-primedRT-IVT method.

[0272] FIGS. 4(A-C) shows the results obtained when the amplificationmethods of the invention were run using a primer comprising a T7 RNApolymerase promoter site and a poly-dT₁₈ sequence (“T7-dT₁₈”), inaddition to using random T7-d N₉ and dN₆ primers. The top graph (A)plots the number of probes (X-axis) versus the log₁₀ (bp) (Y-axis). Themiddle graph (B) plots the number of signatures (X-axis) versus thelog₁₀ (bp) (Y-axis). The bottom graph (C) plots the fraction ofsignatures versus the log₁₀ (bp) (Y-axis). As can be seen in the bottomgraph, the random-primed RT-IVT method helps improve the fraction ofsignificant probes at bp<1000. Using both the T7-dT₁₈ and random T7-dN₉primers for first strand cDNA synthesis improves the fraction ofsignificant probes more efficiently than either the Shannon method orthe method of the invention in which just the random T7-d N₉ primer isused. Stars: Shannon method. Circles: random-primed RT-IVT method.

[0273] These results indicate that the performance of random-primedRT-IVT is stable. The average yield obtained was 20 μg. The protocolproduced little or no 3′ bias and improved the ability to detect the 5′ends of mRNA. Linear amplification extents of 100-fold and labelingefficiencies of approximately 3% can be achieved using this method. Whenpoly-dT and random dN primers, both of which comprise a T7 RNApolymerase promoter sequence, are used together to prime first strandcDNA synthesis, the fraction of significant probes is greater than thatobtained with either the Shannon method or the method of the inventionin which just a random T7-dN₉ primer is used.

[0274] The above results and discussion demonstrate that novel andimproved methods of producing linearly amplified amounts of RNA from aninitial RNA source are provided. The methods of the invention provide animprovement over prior methods of producing linearly amplified RNA inthat the protocol produces little or no 3′ bias and improves the abilityto detect the 5′ ends of mRNA. Furthermore, linear amplification extentsof at least 100-fold can be achieved using the subject methods. Finally,all of the benefits of linear amplification are achieved with thesubject methods, such as the production of unbiased antisense RNAlibraries from heterogeneous mRNA mixtures. As such, the subject methodsrepresent a significant contribution to the art.

[0275] All references cited herein are incorporated herein by referencein their entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

[0276] The citation of any publication is for its disclosure prior tothe filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

[0277] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

1 1 1 36 DNA Artificial Sequence misc_feature 28..36 n = a, t, g, or c 1aattaatacg actcactata gggagatnnn nnnnnn 36

What is claimed is:
 1. A method for amplifying one or more singlestranded nucleic acids, said method comprising: (a) contacting said oneor more single stranded nucleic acids with a first set ofoligonucleotides, each of said oligonucleotides in said first setcomprising a promoter sequence and a sequence from a set of randomsequences of at least 4 nucleotides, a second set of oligonucleotides,each of said oligonucleotides in said second set comprising one of a setof random sequences of at least four nucleotides and one or more enzymesthat alone or in combination catalyze the synthesis of double-strandedcDNA, under conditions suitable for the production of double-strandedcDNA; and (b) contacting the double-stranded cDNA produced in step (a)with a RNA polymerase that recognizes said promoter sequence andribonucleotides under conditions suitable to effect transcription,thereby producing sense or antisense RNA copies corresponding to saidone or more single stranded nucleic acids.
 2. The method of claim 1,wherein the one or more single stranded nucleic acids are poly-A⁺ RNA.3. The method of claim 1, wherein the one or more enzymes is a reversetranscriptase.
 4. The method of claim 3, wherein the reversetranscriptase is rendered incapable of RNA-dependent DNA polymeraseactivity during the transcription step.
 5. The method of claim 4,wherein prior to step (b) said reverse transcriptase is inactivated. 6.The method of claim 5, wherein said reverse transcriptase is inactivatedby heat.
 7. The method of claim 1, wherein a single enzyme is employedfor the synthesis of the double-stranded cDNA.
 8. The method of claim 1,wherein the random sequences of the oligonucleotides in said first setare 6 to 9 nucleotides.
 9. The method of claim 1, wherein the randomsequences of the oligonucleotides in said second set are 6 to 9nucleotides.
 10. The method of claim 1, wherein the random sequences ofthe oligonucleotides in said first set are 9 nucleotides.
 11. The methodof claim 1, wherein the random sequences of the oligonucleotides in saidsecond set are 6 nucleotides.
 12. The method of claim 1, wherein theoligonucleotides in said second set do not comprise a promoter sequence.13. The method of claim 1, wherein each oligonucleotide in said secondset consists of one of a set of random sequences of at least fournucleotides.
 14. The method of claim 1, wherein step (a) furthercomprises contacting said one or more single-stranded nucleic acids witha third set of oligonucleotides each of said oligonucleotides of saidthird set comprising the promoter sequence and a polydT sequence of atleast 5 nucleotides.
 15. The method of claim 14, wherein said polydTsequence is 5 to 25 nucleotides.
 16. The method of claim 15, whereinsaid polydT sequence is 18 nucleotides.
 17. The method of claim 1,wherein the promoter sequence is a T7 RNA polymerase promoter sequenceand the RNA polymerase is T7 RNA polymerase.
 18. The method of claim 1,wherein the ribonucleotides comprise 5-(3-Aminoallyl)uridine5′-triphosphate.
 19. The method of claim 1, wherein the sense orantisense RNA copies are labeled with Cy-NHS.
 20. The method of claim19, wherein the Cy-NHS is Cy3-NHS or Cy5-NHS.
 21. A kit for use inamplifying single stranded nucleic acids into sense or antisense RNA,said kit comprising in one or more containers a first set ofoligonucleotides, each of said oligonucleotides in said first setcomprising a promoter sequence and one of a set of random sequences ofat least 4 nucleotides; and a second set of oligonucleotides, each ofsaid oligonucleotides in said second set comprising one of a set ofrandom sequences of at least four nucleotides.
 22. The kit of claim 21,which further comprises a reverse transcriptase and a RNA polymerasethat recognizes said promoter sequence.
 23. The kit of claim 21 or 22,wherein the random sequences of the oligonucleotides in said first setare 9 nucleotides and the random sequences of the oligonucleotides insaid second are 6 nucleotides.
 24. The kit of claim 21 or 22, whichfurther comprises a third set of oligonucleotides each of which containsthe promoter sequence and a polydT sequence of at least 5 nucleotides.25. The kit of claim 23, which further comprises a third set ofoligonucleotides each of said oligonucleotides of said third setcomprising the promoter sequence and a polydT sequence of at least 5nucleotides.
 26. The kit of claim 24, wherein said polydT sequence is 5to 25 nucleotides.
 27. The kit of claim 25, wherein said polydT sequenceis 5 to 25 nucleotides.
 28. The kit of claim 26, wherein said polydTsequence is 18 nucleotides.
 29. The kit of claim 27, wherein said polydTsequence is 18 nucleotides.
 30. The kit of claim 22 or 25, wherein saidpromoter sequence is a T7 promoter sequence and said RNA polymerase isT7 RNA polymerase.
 31. The kit of claim 24, wherein said promotersequence is a T7 promoter sequence and said RNA polymerase is T7 RNApolymerase.
 32. A method for amplifying one or more single strandednucleic acids, said method comprising: (a) contacting said one or moresingle stranded nucleic acids with a first set of oligonucleotides, eachof said oligonucleotides in said first set comprising a promotersequence and a sequence from a set of random sequences of at least 4nucleotides, and one or more enzymes that catalyze the synthesis offirst strand cDNA, under conditions suitable for the production of firststrand cDNA; (b) contacting the first strand cDNA produced in step (a)with a second set of oligonucleotides, each of said oligonucleotides insaid second set comprising one of a set of random sequences of at leastfour nucleotides and one or more enzymes that catalyze the synthesis ofdouble-stranded cDNA, under conditions suitable for the production ofdouble-stranded cDNA; and (c) contacting the double-stranded cDNAproduced in step (b) with a RNA polymerase that recognizes said promotersequence and ribonucleotides under conditions suitable to effecttranscription, thereby producing sense or antisense RNA copiescorresponding to said one or more single stranded nucleic acids.